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Soil Microbe Lab

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Soil Microbe Lab

Antibiotics are chemicals produced by substances that kill or inhibit the growth of bacterial cells (Hurney et al 2013). These microbes, such as bacteria found in the soil, may seem like they would be harmful to the human body because they attack cells, however they are very efficient at only attacking the bacterial cells. Actinomycetes are one of the more common groups of these soil microbes known to produce antibiotics. Antibiotics work because they target specific aspects found in the prokaryotic cells such as targeting the protein synthesis or preventing the formation of cell walls. In 1928, Alexander Flemming discovered Penicillin on a plate with Staphylococcus bacteria which he was studying and he observed that the Penicillin was inhibiting the growth of the bacteria. What he didn’t know at the time was that he was discovering something that would ultimately act as an antibiotic towards may different kinds of bacteria. However, it was eventually realized that Penicillin works much better on Gram positive bacteria than Gram negative. The reason is that Gram positive bacteria have thicker outer walls which is more easily penetrated by the antibiotic, while Gram negative have thinner walls covered by an extra membrane which acts as a barrier to the antibiotic (Hurney at al 2013). It was later learned that bacteria may also become resistant to antibiotics by mutation in their DNA, or by gaining mutated DNA from other bacterial cells, also called transformation. The objective of this soil microbe experiment is to isolate potential antibiotic-producing microbes in the soil that was taken from a local sample. To do so, two different samples of bacteria have been taken and placed onto agar plates (labelled A and B). As we allow each sample to grow, our objective will be to see what, if any, antibiotics develop and inhibit the growth of the bacteria. We predict that antibiotics will form on the agar plates, thus inhibiting the growth of the E. coli. In this experiment it was our job to isolate potential antibiotic producing bacteria. To do so, we prepared a series of 6 agar plates. For plates 1 and 2, soil samples were taken from random locations around Harrisonburg, sterilized and diluted, and then placed on agar plates and allowed to incubate overnight. This incubation ensured that a variety of soil microbes would be able to grow. (Hurney et al 2013). After soil microbe colonies were allowed to grow, a colony was chosen based on its type of growth -- such as its shape or color -- from each of the given agar plates (1 and 2) and transferred to two new plates labelled A and B. In order to do this transfer, a sterile swab was used to touch only the colony that we wished to transfer to the new plate. We then wiped the swab over the entire surface of the new plate, careful to cover all portions of the plate. Plates A and B were then allowed to incubate until the desired amount of growth had occurred (Hurney at al 2013). After this given amount of time had elapsed, a new plate, Plate 5, was then used. Plate 5 was divided into four sections, with two of the sections labelled A and two of the sections labelled B. Using a pipettor set, 100µL of a liquid culture of Escherichia coli was deposited into the center of the plate and then spread to cover the entire surface. A sterile straw was then used to remove a chunk of bacteria from Plate A, which was then placed into the appropriate section A on Plate 5. Using a new sterile straw, another circular chunk of bacteria was removed from Plate A and placed on the remaining section A on Plate 5. For Plate B the same steps were repeated and the bacterial discs placed on the appropriate sections labelled B on Plate 5 (Hurney et al 2013). Plate 6 was the last plate we prepared. To do so, we divided the agar plate into 3 sections, which would be the test site one of three known antibiotics: Tetracycline, Gentamicin, and Penicillin. Once the plate was correctly divided, 100µL of the E. coli liquid culture was then deposited into the center to the agar plate and spread to cover the entire surface. The three antibiotic discs were then placed in their appropriate sections on the agar plate (Hurney et al 2013). Plate 6 could then be used to interpret the results of Plate 5 by offering an explanation of why the outcome of Plate 5 occurred, whether it be that antibiotic producing bacteria were isolated or not. Once all 6 plates were prepared, the growth was then monitored. For growth plates 1 and 2, we saw thick growth with a wide variety of bacterial colonies. A larger, cream colored colony of irregular shaped bacteria was found to dominate most of Plate 1. A filamentous yellow colored colony was also present on this plate. On Plate 2, a small, yellow cocci shaped bacterial colony was found growing sporadically across the agar plate. A sample of the irregular shaped bacteria was placed on Plate A and allowed to grow. We found that the colony grew rapidly, which resulted in thick, yellow colored growth of the bacteria. A sample of the filamentous bacteria was also taken and placed on Plate B. When allowed to incubate, the results were much the same as Plate A. The growth was extremely thick and yellow in color. Once the plugs from these two plates were transferred to the experimental plate (Plate 5), the colonies were then allowed to grow in order to determine whether or not any zones of inhibition would be formed. We determined that zones of inhibition were not formed for any of the samples, however the growth for each sample did differ. Sample A thrived in the conditions much better than did Sample B, with growth plug diameters exceeding 20mm for both samples, whereas the growth plug samples for Sample B did not exceed 10mm for either sample. For the Antibiotic Plate (Plate 6), the results were much different. Zones of inhibition were found around both the Gentamicin and the Tetracycline antibiotic discs. The Tetracycline antibiotic disc had the largest zone of inhibition, with the diameter reaching 24mm. Gentamicin had a zone of inhibition of about 10mm. The last antibiotic disc, Penicillin, did not have a measurable zone of inhibition. For this experiment, we hypothesized that the samples taken from Plates A and B would produce antibiotics. We predicted that these antibiotics would be effective against E. coli, thus producing a zone of inhibition around the samples. However, our findings were not consistent with our prediction, and it was unable to be determined whether antibiotics were produced or not. This is because the bacteria could possibly have been Gram-negative, which would not cause a zone of inhibition to be formed because the bacteria would not be inhibited from growing. The E. coli could also have a high resistance to the bacteria, which does not necessarily mean that no antibiotic was formed, just that the antibiotic was ineffectual towards the E. coli. The last alternative explantation for the lack of zones of inhibitions found is that the bacteria just was not producing antibiotics. Based on these alternative explanations, we cannot entirely reject our hypothesis because it may have just been the genetic makeup of bacteria we chose to use, whether it be that the bacteria was Gram-negative or that the antibiotic just was not strong enough to combat the growth of the E. coli. If another experiment was conducted, simply choosing different bacteria samples could be enough to provide evidence to support our hypothesis of antibiotic production.

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