Control by Antibiotics and Antiseptics Lab Report
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The fact that each bacterial species, and even some of their respective strains, responds uniquely to a given antimicrobial makes it necessary to have methods, which provide researchers and clinicians with measurable susceptibilities. This need has become prevalent since the rising of later generations of antimicrobial compounds achieved by chemical modifications of the naturally occurring antibiotics. By utilizing the Kirby-Bauer method, researchers and clinicians are able to determine the susceptibility of given microorganisms to different antibiotics and antimicrobials. While antibiotics specifically target bacteria, antimicrobials cast a wide net, nonspecifically targeting microorganisms and viruses. During this lab, we tested the effects of both antimicrobials and antibiotics on different bacterial cultures. By creating different lawns of bacteria and placing on each of them disks soaked in different antibiotics and antimicrobials, we were able to observe the susceptibility of the differing bacteria to several agents. This process that we practiced is the Kirby-Bauer method. As stated before, antibiotics and antimicrobials serve differing purposes. Antibiotics are substances, usually in the form of medication, that destroy bacteria or inhibit the growth of bacteria.
On the other hand, antimicrobials act in destroying or inhibiting the growth of all microorganisms. One type of antimicrobial is an antiseptic, which acts to reduce the presence of microorganisms without harming living animal tissue (El-Mayas, 2013). It is important to note that while we were able to measure the susceptibility of these agents, we cannot conclude that resistance is present because further investigations would have to be conducted to determine if the microorganism has developed a metabolic capability to withstand the agent’s mechanisms and effects (Maxwell, 2013). We observed the effects of several different antibiotics and antimicrobials on four different bacterial cultures. The four bacteria tested were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Saccharomyces cerevisiae. On each bacterial culture, penicillin, streptomycin, tetracycline, chloramphenicol, erythromycin, and vancomycin were inserted on disks by utilizing a plunger.
Penicillin interferes with peptidoglycan synthesis by inhibiting penicillin-binding proteins. Penicillin is bactericidal and most effective against gram positive bacteria. Streptomycin are bactericidal against aerobic and facultative bacteria by binding to the 30S subunit of the ribosome, which effectively blocks initiation of translation and causing the mRNA to be misread. The tetracycline is bacteriostatic against many gram positive and negative bacteria because it also binds to the 30S subunit, blocking the attachment of tRNA. Chloramphenicol is bacteriostatic and broad-spectrum; it binds to the 50S subunit and prevents the formation of peptide bonds. Erythromycin prevents the continuation of protein synthesis by binding to the 50S subunit and is bacteriostatic against many gram positive bacteria. Lastly, vancomycin is bactericidal against gram positive bacteria because it blocks the synthesis of peptidoglycan chains.
Vancomycin and chloramphenicol are only utilized by medical personnel as a last resort to treat life-threatening infections (El-Mayas, 2013). Based upon the differing mechanisms of each antibiotic, we expected the results to be vast for each in regards to each bacterial culture. In addition to observing the susceptibility to the antibiotics, we also observed the susceptibility to five types of antimicrobials. We chose to infiltrate our plates with Lysol, bleach, betadine, glycerol, and hydrogen peroxide. Lysol is used in lower concentrations as a household disenfectant and utilizes phenolic compounds to destroy cytoplasmic cell membranes and denature proteins.
Bleach is also used as a household disenfectant, whose chlorine solutions react with organic compounds, resulting in a germicidal effect. Betadine is an idophor, which is used most frequently as an antiseptic and its exact mechanism is still unclear. Glycerol is a sugar alcohol that is often used in soap-making. While glycerol does have some antiseptic properties, it is primarily used for preservation or as a sweetener. Lastly hydrogen peroxide is used for the debridement of wounds because it creates free radicals, which damage the organic compounds. Because antimicrobials often have a more generalized effect, we expected the results to be more consistent than the antibiotics, but still varied. Methods
In order to observe the different effects of antibiotics and antimicrobials on different bacterial cultures, we utilized the Kirby-Bauer method. With four different bacterial species and two plates for each, one to test antibiotics and the other to test antimicrobials, we created lawns on each plate with the respective bacteria. Before creating the lawns, one must label each plate to indicate the sample, the method, and the types of antibiotics or antimicrobials used. To create a lawn, one sterilizes an inoculating loop with an open flame, allows it to cool, dips it into the sample, and then proceeds to create a dense zig-zag pattern that covers the area of the plate in one direction. Then the plate is rotated 45 degrees and the process is repeated. Finally the plate is rotated 45 degrees once more and the process is repeated again. The creation of a lawn is performed on each plate with the respective samples of bacteria. After this, a disk dispenser containing several disks of differing antibiotics is plunged into four plates of each of the different bacteria species. Results
Diameter of Zone of Inhibition in milimeters (mm)
E. coliS. cerevisiaeP. aeruginosaS. epidermisANTIBIOTIC penicillin60 0 0 0 streptomycin100 0 40 40
tetracycline20 0 0 120
chloramphenicol220 0 0 160
erythromycin240 0 10 20
vancomycin100 0 0 0
ANTIMICROBIAL lysoltoo large too large 0 140
bleachtoo large too large 380 0
betadinetoo large too large 50 40
glycerol0 0 0 0
hydrogen peroxide too large too large 200 160 for the antibiotics: red=possible resistance orange=intermediate yellow=susceptible white=not specified/susceptible
El-Mayas, H. (Presenter). (2013, September 26). Humans of the microbial world. Lecture presented at Georgia State University, Atlanta, GA. Maxwell, R. (2013). Control by Antibiotics and Antiseptics. Retrieved October 10, 2013, from http://www.bologsu.us/BOLO/course/view.php?id=3§ion=7