Bacteria are microbial organisms which are present in various environments. Many bacteria are good and help humans synthesize materials and assist in biological processes, such as digestion. However, some bacteria can cause harmful diseases. When harmful bacteria infects the body, people take antibiotics in order to suppress bacteria. Different antibiotics have different ways they suppress bacteria, and the efficacy of antibiotics depends on the structure of the bacteria which they are targeting. The specific bacteria being studies in this experiment is E Coli and Enterococcus.
E Coli is a bacteria which may cause diarrheal disease (CDC. ov), and Enterococcus may cause urinary tract infection (Kau). Bacteria are primarily classified as either Gram Positive or Gram Negative. Gram Positive Bacteria lack an outer membrane and have multiple layers of peptidoglycan than can range from 30nm-100nm, whereas gram negative bacteria have an outer membrane and only a few layers of peptidoglycan, which are a few nanometer thick (Silhavy, 2010). Different antibiotics can affect gram positive and gram negative bacteria to different degrees. The experiment done consisted of testing how different antibiotics affect different bacteria.
The independent variable during this experiment would the radius of the zone around the antibiotic which would not have any bacteria present. The dependent variable is which antibiotic, if any, was placed near the bacteria. The control was the bacteria which did not have any antibiotic placed near it. The hypothesis was that each antibiotic would affect each bacteria to the same degree, and the predicted outcome of this trial was that the bacteria would all be sensitive with a zone size of about 10 mm, since that is where the cutoff of sensitive to very sensitive was made.
Vancomycin, Ampicillin, and Tetracycline antibiotic disks were used in the experiment. Ampicillin was used because of its comparatively similar structure to penicillin (Raynor). Ampicillin is just a penicillin molecule with an extra amino acid added to it. Since penicillin is often used in modern medicine, and is effective, one could reasonably think than Ampicillin would be fairly effective as well. Tetracycline was used because it is an antibiotic used to treat urinary infection (Nlm), and if so, then it might be effective against Enterococcus.
Vancomycin was used in the experiment because it is considered a drug of last resort (Boneca), and the experiments wished to know how effective a drug of last resort could be. MATERIALS AND METHODS: Before the actual experiment can be carried out, the Gram values of the bacteria need to be analyzed. A small drop of water is placed on a clean slide. And a swab is used to take some E Coli, and that E Coli is spread into the water. Once the slide is dry, it is quickly passed over the flame of a Bunsen burner. Then, the slide is washed with water.
A few drops of crystal violet is placed on the slide, and then water is used to rinse the slide. Then, the smear is covered with Gram iodide for one minute. The slide is once again rinsed with water. Destaining is done by dropping 95% alcohol/acetone mixture down the slide, drop by drop, until only a faint violet color is seen. Then the slide is rinsed immediately, and later safranin 30 to 60 seconds. The slide is then rinsed with water, blotted with bibulous paper. Finally, a coverslip is placed on the slide, and the slide is examined in the microscope.
Gram positive bacteria should look purple, and gram negative bacteria should look pink. Six clean and dries 10 cm diameter agar plates were gathered, and labelled with a sharpie such that each plate was divided into four segments, each labeled from zero to three, and each plate contained the name or initial of the experimenters. In order to prepare a bacterial lawns of E Coli, a sterile swab was inserted into an already growing bacterial culture, and the agar plate was covered with E Coli. An antibiotic disk of Vancomycin was placed in the quadrant of the with “1” written on it.
An antibiotic disk of Ampicillin was placed in the quadrant of the with “2” written on it. An antibiotic disk of Tetracycline was placed in the quadrant of the with “3” written on it. Two more ar plates with E Coli were made, using the previously stated instructions, for a total of three agar plates with E Coli. The entire process was repeated, but instead of E Coli, the new agar plates contained Enterococcus. When all 6 agar plates, three Coli and three with Enterococcus were created, were then placed in an incubator at 37 C for a week. The agar plates were then removed, and analyzed, in order to reach a conclusion.
RESULTS: As seen in figure 2, bacteria which was not exposed to antibiotics kept growing, and Vancomycin had no effect on E Coli. Vancomycin did, however, have an effect on Enterococcus. Ampicillin and Tetracycline both effects on Enterococcus and E Coli. Table 1 contains the data in numerical format. Table 1 Bacteria Gram +/- Average Zone with no antibiotic (Control) (mm) Average zone with antibiotic Vancomycin (mm) Average zone with antibiotic Ampicillin (mm) Average zone with antibiotic Tetracycline (mm) Enterococcus Positive 0 12 20 14 E Coli Negative 0 0 15 20
Figure 1* The image on the eft is an example of gram-positive bacteria, and the image on the right is an example of gram negative bacteria. *- The experiments neglected to take pictures of the stained slides, so Figure 1 was gathered from an outside source and was similar to the results of the gram staining. Credit: Center for disease control, National Institute of Allergy and Infectious Diseases Figure 2. Figure 3: E Coli Enterococcus
DISCUSSION AND CONCLUSION: The overall consensus from the experiment was that Vancomycin was the least effective antibiotic for both the Gram Positive and Gram Negative strains, Ampicillin was the most effective on the Gram Positive Strain, and Tetracycline was the most effective on the Gram Negative Strain. The control showed no signs of bacterial death, as was expected. The hypothesis and prediction were both incorrect, and so more research was done in order to understand why different antibiotics worked differently on different bacteria, and to observe if any of the results indicated a new aspect of the antibiotic-bacteria relationship or reinforced prior norms.
The fact that Vancomycin was completely ineffective against E Coli was quite surprising. The explanation of why Vancomycin was ineffective in the Gram Negative Bacteria is that Vancomycin is a glycopeptide antibiotic (Van). Glycopeptide-based antibiotics are not effective against gram negative bacteria, since the large Glycopeptide molecules cannot penetrate the outer membrane of Gram Negative Bacteria” (French, 1998). Since Vancomycin could not enter the bacteria, it had no effect on bacterial picillin was effective against E Coli and Enterococcus, but it was more effective against Gram Positive Endrofaucus than gram Negative E Coli.
The reason why that was so was because Ampicillin functions by binding to penicillinbinding proteins (PCP) and preventing cell wall synthesis (Adnan,). Since PCP is used to create peptidoglycan (Scheffers), and Gram Positive Bacteria has more peptidoglycan than Gram Negative bacteria (Silhavy, 2010), it can be reasonably assumed that Gram Positive Bacteria such as Enterococcus has a high concentration of PCP than Gram negative bacteria such as E Coli. Since Enterococcus has a greater concentration of PCP than E Coli, Ampicillin is more effective on Enterococcus than on E Coli.
Tetracycline was more effective against E Coli than Enterococcus. The reason for this increased effectiveness could be the rate of antibiotic absorption. Tetracycline goes into bacteria through, most likely, passive transport (Schnappinger), and Gram Positive bacteria have a much thicker wall than the Gram Negative Bacteria (Silhavy, 2010). So it makes sense that Gram Negative E Coli would be able to absorb tetracycline at a faster rate than the gram positive Enterococcus, since gram positive has a thicker, less permeable cell wall than gram negative E Coli.
One problem with the study was the small sample size. If more agar plates were made, then it would be easier to see trends in the results. Also, a larger sample size consisting of different bacteria cultures would decrease the likelihood than a certain colony contained mutations which make them more resistant to antibiotics. Most of the data did was precise, in that results of the trials with the same conditions were similar with no statistical anomaly. This indicates that no significant error was committed.
These results falsify the hypothesis that all antibiotics would affect all bacteria equally, and lend credence to the idea that some antibiotics are more effective against some kinds of bacteria than others. Once again, more tests must be done to reinforce these results. More testing must be done to see how various bacteria react to various antibiotics, in order to determine the most effective course of treatment for various bacteria caused illnesses. A possible future study could involve examining rates of efficacy.
If the agar plates had been examined every day that would showed information about the rate of antibiotic effectiveness. A future possible study would involve more than just one kind of Gram Negative and Gram Positive bacteria. If the antibiotics react to various kind of gram negative and gram positive bacteria as they did in this experiment, it would help establish that the difference in the bacteria’s out wall, it gram negative/ gram positive aspect, and the effectiveness of the antibiotic had a causational relationship, instead of just a corollary one.
These are options for future studies. None of these trials can be used as foolproof evidence about the interaction between antibiotics and bacteria. However, this experiment does show that since different antibiotics affect different bacteria if different ways, antibiotics which destroy harmful bacteria while keeping helpful bacteria alive could lead to more efficient treatments. As with any experiment, more trials and experimentation must be done to arrive at a sound conclusion which could be applied to help humanity.