60 Petri Dish
The ten Petri dishes that exclusively did not contain an antibiotic in the bacteria culture served as the control. There were ten trials for the control and each level of IV. The experiment began by cleaning the work area and sterilizing it with 70% ethanol. Then trypticase soy agar (TSA) was poured into six groups of 60 Petri dishes (See Appendix 1). The dishes were labeled based on the antibiotic used and were left to dry and solidify at room temperature. After an hour, the dishes were placed in a refrigerator at three degrees Celsius. If there was visible condensation on the agar plate, they were flipped to avoid the contact of heat that drove the moisture out of the agar dishes. The contents of the broth culture of E. coli was gently swirled
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until it was equally murky throughout in the test tube. Five glass rods and 60 sterile cotton swabs were used to create a lawn of E.
coli on the plates of TSA. The swab was dipped into the test tube containing the E. coli bacteria culture and was swiped delicately over the agar surface of the 60 Petri dishes (See Appendix 2). In order to ensure that the entire surface area of the dish was covered, the E. Coli bacteria culture was swabbed in at least 3 different directions. The different types of antibiotics were applied to each of the agar plates by using five sterile forceps for each antibiotic. The forceps were cleaned with the ethanol disinfectant before using it again for another Petri dish. The forceps were used to pick up the 60 antibiotic discs and dip them into their corresponding antibiotic solution. This ensured that the IV would receive the same amounts of the solution for each of the Petri dishes. The antibiotic discs were placed at the center of the Petri dish according to their labels and the plates were wrapped with Parafilm. The dishes were incubated for 24 hours at 37 degrees Celsius. The procedure allowed for the IV to be controlled because of the incubation. This allowed the Petri dish to stay sterile and also protected against foreign bacterial exposure during the
incubation. After 24 hours, the Petri dishes were removed from the incubator and were inspected for antibiotic activity. To calculate and measure the antibiotic activity, the Kirby-Bauer disc diffusion antibiotic sensitivity test was used. Each of the Petri dishes were inspected for an inhibition zone, the area around the antibiotic disc where there is no bacterial growth (See Appendix 3). This accurately and effectively measured the susceptibility of a bacteria to an antibiotic because as the antimicrobial diffused out from the disc into the agar, there was a higher concentration nearer the disk. The concentration decreased gradually, as the distance from the edge of the disc increased. As long as the concentration of antibiotic at any given distance is high enough to properly act as an antibacterial agent, no bacteria will grow at that location. Hence, if the bacteria have increased resistance, a smaller inhibition zone would be observed, and vice versa. The average class data for the diameters of the zones of inhibition were recorded in a lab notebook. The adult mentor of this experiment provided safety goggles, safety gloves, and a lab apron that was worn when handling the bacteria. All bacterial cultures were treated as potential hazards and tubes, pipettes, or other tools that come in contact with bacterial cultures were treated with care, in order to avoid ingesting or inhaling any bacteria. Ethanol also releases harmful vapors, so the experiment was performed in a well-ventilated room with windows open. Soap and water were used to cleanse after handling the bacteria. Bacterial cultures, plates, and disposables that were used to manipulate the bacteria were soaked in a 10% bleach solution (one part bleach to nine parts water) for one to two hours. At the end of the experiment, 70% ethanol was used as a disinfectant to thoroughly clean any surfaces that were used. All materials that came in contact or were used during the bacterial phase of the experiment were disposed of in a biohazardous bag and given to the adult mentor to be discarded. The same steps for the other antibiotics were repeated. After all 60 trials, data and observations were recorded.
until it was equally murky throughout in the test tube. Five glass rods and 60 sterile cotton swabs were used to create a lawn of E.
coli on the plates of TSA. The swab was dipped into the test tube containing the E. coli bacteria culture and was swiped delicately over the agar surface of the 60 Petri dishes (See Appendix 2). In order to ensure that the entire surface area of the dish was covered, the E. Coli bacteria culture was swabbed in at least 3 different directions. The different types of antibiotics were applied to each of the agar plates by using five sterile forceps for each antibiotic. The forceps were cleaned with the ethanol disinfectant before using it again for another Petri dish. The forceps were used to pick up the 60 antibiotic discs and dip them into their corresponding antibiotic solution. This ensured that the IV would receive the same amounts of the solution for each of the Petri dishes. The antibiotic discs were placed at the center of the Petri dish according to their labels and the plates were wrapped with Parafilm. The dishes were incubated for 24 hours at 37 degrees Celsius. The procedure allowed for the IV to be controlled because of the incubation. This allowed the Petri dish to stay sterile and also protected against foreign bacterial exposure during the
incubation. After 24 hours, the Petri dishes were removed from the incubator and were inspected for antibiotic activity. To calculate and measure the antibiotic activity, the Kirby-Bauer disc diffusion antibiotic sensitivity test was used. Each of the Petri dishes were inspected for an inhibition zone, the area around the antibiotic disc where there is no bacterial growth (See Appendix 3). This accurately and effectively measured the susceptibility of a bacteria to an antibiotic because as the antimicrobial diffused out from the disc into the agar, there was a higher concentration nearer the disk. The concentration decreased gradually, as the distance from the edge of the disc increased. As long as the concentration of antibiotic at any given distance is high enough to properly act as an antibacterial agent, no bacteria will grow at that location. Hence, if the bacteria have increased resistance, a smaller inhibition zone would be observed, and vice versa. The average class data for the diameters of the zones of inhibition were recorded in a lab notebook. The adult mentor of this experiment provided safety goggles, safety gloves, and a lab apron that was worn when handling the bacteria. All bacterial cultures were treated as potential hazards and tubes, pipettes, or other tools that come in contact with bacterial cultures were treated with care, in order to avoid ingesting or inhaling any bacteria. Ethanol also releases harmful vapors, so the experiment was performed in a well-ventilated room with windows open. Soap and water were used to cleanse after handling the bacteria. Bacterial cultures, plates, and disposables that were used to manipulate the bacteria were soaked in a 10% bleach solution (one part bleach to nine parts water) for one to two hours. At the end of the experiment, 70% ethanol was used as a disinfectant to thoroughly clean any surfaces that were used. All materials that came in contact or were used during the bacterial phase of the experiment were disposed of in a biohazardous bag and given to the adult mentor to be discarded. The same steps for the other antibiotics were repeated. After all 60 trials, data and observations were recorded.