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Examining the Effects of Antibiotics and Antibacterials on Bacteria Introduction Bacteria are prokaryotic organisms that have one circular chromosome and can divide to produce a new generation of bacteria every 20 minutes if they have enough food and resources. Even though they reproduce asexually and have to rely mostly on mutations for new genetic combinations, their rapid reproductive rate allows for rapid adaptation to a changing environment. Bacteria have traditionally been divided into two broad groups – gram positive and gram negative. This was based on their reactions to different antibiotics. Today, because natural selection has acted on (and continues to act on) bacteria, favoring traits that make bacteria better able to survive their environments (i.e. be resistant to antibiotics), these traditional groupings are no longer relevant as the antibiotics that were once widely used are less and less effective. Over-the-counter medications and soaps advertise their ability to kill "germs" (bacteria). Most soaps and cleaning agents today claim to be "antibacterial." Technically, antibacterial substances used on living organisms are called antiseptics and those used for cleaning surfaces are called disinfectants. Studies are showing that many common bacterial strains are adapting to these over-the-counter products and becoming resistant to them. Traditionally it made a difference in how you treated a bacterial infection depending on whether bacteria were gram positive or gram negative. With widespread antibiotic resistance in the bacteria that cause disease, the distinction between gram positive and gram negative is not as important when we are forced to use our strongest and most sophisticated antibiotics. That said, we have strains of harmless gram positive and gram negative bacteria that we will use to examine the effects of various antibiotics and antibacterial on different bacteria. E. coli is a gram negative bacteria normally found in the large intestine of people. They help us absorb water and secrete vitamin K. B. cereus are gram positive soil bacterium. Bacteria are cultured or grown on plates (in agar media) and in culture tubes (in liquid media) for study in the laboratory. Using sterile technique, we will culture our E. coli and B. cereus on LB agar plates and observe the effects of antibiotics and antibacterials on each kind of bacteria. The sterile technique we will use will help us prevent contaminating our bacterial cultures and ourselves. Materials (per group) cell spreader(1) permanent marker (1) Bunsen burner (1) Sterile, disposable 1ml pipette (4) 10% bleach solution waste beaker LB agar plates (4) various antibiotic disks blank disks flint various antibacterials E. coli culture B. cereus culture Procedure (Day 1: Setting up the Plates) Getting Started 1. Leave all of your personal items on your desk. You should NOT have any of your personal belongings (backpacks, sweatshirts, etc.) on the laboratory table during this investigation!! 2. Use the spray bottle with 10% bleach solution to wipe down your table. 3. Obtain stock culture bottles of E. Coli and B cereus. Be careful when handling these! Do not open them until you are ready to use them! 4. Obtain 4 nutrient agar plates. Be careful when handling these as they are sterile!!
5. Organize the supplies at your table. Labeling the Plates 1. Write your period and lab group on the bottom of each of your LB agar plates. 2. On all of the plates, draw intersecting lines on the bottom of the dish so that it is divided into quarters (see picture below):
a,. On two of the plates, write E. coli. You will spread E. coli cells onto this plate. b. On t he other two plates, write B. cereus. You will spread B. cereus cells onto this plate. Transferring your Cells 1. Light your Bunsen burner and adjust the valves on the bottom of the burner as needed to ensure a good flame. If you need help lighting your Bunsen burner, ask. Do NOT fool around with the gas! Any horseplay will result in a zero on this lab, a referral, and a trip to the Associate Principal! 2. You are going to spread 1 ml of cells from the E. coli culture bottle onto each of the two plates you have labeled E. coli. Here is the procedure for spreading cells: (1) Remove the lid of the E. coli culture, press the bulb on the pipette and then insert the tip into the E. coli suspension (2) Release the bulb of the pipette and withdraw 1 ml of the E. coli suspension (the line right below the bulb on the pipette) (3) Open the plate marked "E. coli" and depress the bulb of the pipette to expel the E. Coli suspension onto the top of the agar. (4) Place the pipette in the waste beaker. (5) Pour the ethanol from the culture tube into the petri dish Dip the spreader into the petri dish with ethanol and briefly pass it through the Bunsen burner flame to ignite the alcohol. Allow the alcohol to burn off away from the Bunsen burner flame. The spreader will be too hot if you leave it in the flame. Do not play with the alcohol and flame!! (6) Lift the lid of one of the E. coli plate enough to allow spreading; do not place the lid on the lab table! (7) Cool the spreader by gently rubbing it on the surface of the agar away from the cell suspension (8) Touch the spreader to the cell suspension and gently drag it back and forth several times across the surface of the agar. Rotate the plate one quarter turn and repeat the spreading motion. Be careful not to gouge the agar. (9) Replace the lid. Return the cell spreader to the ethanol without flaming (10) Set the plate aside for now. (11) Repeat this procedure for your second E. coli plate. 3. Now take the plates labeled B. cereus and spread the B. cereus cells from the B. cereus bottle onto these plates, repeating the procedure for spreading cells outlined in steps 1-11 above using a fresh 1 ml pipette. Transferring Antibiotic Disks 1. You are now going to add antibiotic disks one of the E. coli and one of the B. cereus plates you just
spread. You will be transferring 4 disks to each plate – one disk in each quadrant. You are going to add the same four antibiotic disks to each plate in the same quadrant (1, 2, 3, and 4). Use the antibiotics that are at your table. What four antibiotics are you using? Antibiotic in quadrant 1: Antibiotic in quadrant 2: Antibiotic in quadrant 3: Antibiotic in quadrant 4:
2. The sketch below gives you an idea of where to place each disk. Use the dispenser to carefully place each antibiotic disk on the two plates. If a disk does not go on exactly the right location, sterilize your forceps in the Bunsen burner and use them to move the disk to the correct spot on the plate.
Transferring Antibacterials 1. You are now going to transfer four different antibacterials to your other E. coli and B. cereus plates. 2. Before you start, record which antibacterials you are using below. You will dip blank disks into each antibacterial and put the disk on your plate in one of the quadrants like you did with the antibiotic disks. The same antibacterial needs to be in the same quadrant on each plate. What four antibacterials are you using? Antibacterial in quadrant 1: Antibacterial in quadrant 2: Antibacterial in quadrant 3: Antibacterial in quadrant 4: 3. Sterilize your forceps by running them through the flame, and then use them to pick up a blank disk, dip it into one of your "antibacterials" and then place the disk in the appropriate quadrant on your E. coli plate. a. Repeat the procedure for the same antibacterial onto your B. cereus plate. b. Repeat the procedure for the rest of your antibacterials and place these disks onto the appropriate quadrants that you identified in #2 above. After the Transfers 1. The cells need to soak in on the plates for a while so leave them, face up, on your tables as you clean up the rest of your supplies.
2. Turn off your Bunsen burner, and return your supplies to where you got them. Carefully bring the stock culture bottles of E. Coli and B. cereus up to the teacher demonstration table. Make sure you keep the lids on!! 3. Wash your hands with soap. After you have put your plates in the incubator (Mr. Stewart will tell you when to do this), you will need to re-bleach your table. Procedure (Day 2: Observing, Sharing Data, and Clean Up) 1. Obtain your plates. 2. Observe the two plates that had antibiotics on them. Record your results below and add your results to the class data sheet. 3. Observe the two plates that had antibacterials on them. Record your results below and add your results to the class data sheet. 4. Copy down the class data as you will need this to answer the lab questions.