Differential Staining Lab Report

This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.

E.coli outbreaks have steadily grown over the last few decades. An expansion in big farming has led to E. coli not only being found in meat, but vegetation as well, due to waste runoff. This has increased our need for adequate antibiotics that can fight bacteria, like E. coli. The best way to pinpoint which antibiotics work is by measuring their ability to create antimicrobial agents or zones of inhibition. When a paper disc that has been saturated in an antibiotic is inserted in a solution of E.coli and medium, the zone of inhibition will be noted as the clear ring that forms around the disk. The antibiotics efficacy is then determined by measuring each disk zone of inhibition, and comparing these measurements to the zone measurements of an untreated specimen. If an antibiotic is to be deemed sufficient for treating E. coli it should show a zone of inhibition that is at least double the size of the untreated specimen.

Whole mount preparations were prepared using nerves which were harvested from animals sacrificed using Ketamine 40-100 mg/kg IP and Xylazine 5-13 mg/kg IP. Animals underwent transcardial perfusion with 500 ml of 0.9% saline, followed by 500 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer.

Coli. The first standard E. Coli has no resistance plasmid while the second strain contains a resistance plasmid with genes protecting it from ampicillin. This standard E. Coli and pAMP (plasmid-Ampicillin) E. Coli were each streaked across plates containing the antibiotic and containing growth supportive Lurithea Broth. The purpose of this lab was to test their growth in each medium. Our hypothesis was that while the ampicillin resistant E. Coli would show growth in both LB and LB-AMP plate, the standard E. Coli would only grow in the LB plate for it contains no resistant plasmids against the

b.Describe how the organism would look if it is stained with nigrosin. How does this stain interact with the cell?

Antibiotic resistance is when microorganisms, such as bacteria, are able to survive an exposure to antibiotics and these bacteria are now resistant to the effects of these antibiotics. Antibiotic resistance in bacteria has been an issue since antibiotics were discovered. The fact that bacteria can become resistant to our medical treatments such as antibiotics is a natural evolutionary process, but there are certain human contributions that definitely speed up the process. For example, one of the main contributions that will be discussed is the problem of over prescription of the antibiotic drugs. The

After analyzing the data recorded for both the agar plates containing ampicillin and those that did not, it can be concluded that the data provides enough evidence to reject the null hypothesis. There is enough evidence to support the alternative hypothesis stating that there is a correlation between plasmids coding for an antibiotic resistant gene and bacterial growth in ampicillin. When a bacterial solution containing either pUC18 or the lux plasmid is transformed in an agar plate containing ampicillin, only those cells which took in the plasmid are able to survive and replicate, forming individual colonies. Not all cells are transformed though, the chances of a successful transformation were extremely low. In order to see which cell transformed the cells were tagged according to their plasmid, in the plates containing pUC18 the only

When antibiotic is used most of the bacteria die but a few bacteria with antibiotic resistance gene survive and reproduce and pass this advantage to their offsprings. This selective pressure exists naturally, however antibiotic misuse can be accused for fastening the spread of the antibiotic resistance gene [Refer to figure 2] (Learn Genetics 2015). Consequently, inappropriate antibiotic intake will lead to a greater chance of superbugs being developed. Antibiotic resistance can be defined as a new ability which a bacterium has developed to stay unattached in the presence of an antibiotic that was previously effective to destroy the bacterium (ABC science 2015). Four key mechanisms that has been identified for bacterial antibiotic resistance can be listed as: producing enzymes that inhibit the functionality of the drug, reducing the effectiveness of the drug by producing targets against which the antibiotic, reducing the permeability of the drug into the bacterium and active export of antibiotics using various pumps (Pogson 2012). All these mechanisms can be developed by any of the bacteria when the corresponding mutated gene of antibacterial resistance is received. The genes code for specific proteins, and variation in the gene leads to alteration of the shape of proteins. This leads to changing the functionality

According to a study conducted in a Tunisian Hospital, E.coli resistance rate to ampicillin was 72.1% (Ferjani et al., 2014). Furthermore, penicillin was found to be least effective against E.coli with a 100% resistance rate during a research done in trying to comprehend the patterns of antibiotic susceptibility of bacteria (Saba et al., 2014). E.coli found in the intestinal contents of pigs was found to be 57.7% resistant to streptomycin (Lee et al., 2014). Situated on these past research and studies, if a tryptic soy agar plate in applied with E.coli and separated into four different quadrants with three quadrants having an antibiotic disk and one having the control disk, then the results will show E.coli to be most resistant to penicillin, somewhat resistant to ampicillin and least resistant to

The purpose of this experiment is to show how different concentrations of Ampicillin affect Escherichia Coli Growth and how the bacteria become resistant to the antibiotic drug. Through a series of steps, which involves streaking agar plates with E.coli sample and application of ampicillin to the E.coli sample on the agar plate, the experiment yields a result that supports the hypothesis. The hypothesis acclaims that ampicillin would affect the growth of E.coli; measuring the zone of inhibition approves the claim in the experiment. The measurements of the zone of inhibition indicate that the generation of E.coli expands as the radius reduces. The reduction of the radius shows the E.coli population is reducing and becoming resistant. In other

Aim – To test the effectiveness of various anti-microbial substances on the growth of a bacteria called Escherichia coli, also known as E. coli.

Results of the “-DNA” plate coincide with the expectation of lawn growth due to the non-selective Luria broth and agar plate which allows for almost anything to grow. The “-DNA/+AMP” labeled plate also yields expected results with no bacterial cell growth. The cells do not contain DNA plasmid that encode for the antibiotic resistant gene, preventing the formation of the bacterial cell wall. There is a discrepancy in the agar plate labeled “+DNA/+AMP” because a blue colony is observed and the plate contains bacterial cells. This unexpected growth could be due to cross contamination with the “+DNA/+AMP/+IPTG” plate. For the last plate (+DNA/+AMP/+IPTG), the observations solidify the expectancy of minimal bacterial growth with production of pigmentation. One colony is observed with satellite cells, in addition to a miniscule spot of blue pigmentation. The “Competent” cells are capable of resisting ampicillin. IPTG, the inducible promoter, results in the production of color. With gene regulation, “competent” cells can acclimate to their environment due to methods of turning genes on and off5.

Three of the four quadrants were labeled S, P, T, for streptomycin, penicillin, and tetracycline. One quadrant was left blank for the control (Figure 1.1) Having a control of no antibiotic was used to show the growth of the bacteria when it is uninhibited by antibiotics. Then, Escherichia coli K12 from VWR (catalog #470179-082) and Staphylococcus aureus from VWR (catalog #470179-208) were evenly swabbed across the surface of the agar plate with a cotton swab; ten plates, contained E. coli and the other ten plates contained S. aureus. Then, Streptomycin (10 μg/mL), Penicillin (10 U = ~10 μg/mL), and Tetracycline (30 μg/mL) were obtained and placed into the middle of their respective quadrants. After this, the agar plates were stacked into two stacks of five, for each bacteria, were wrapped with parafilm, and were incubated at 37°C for 48 hours (Figure 1.2). After 48 hours, the plates were moved to a refrigerator at 4°C, until they were taken out of the fridge approximately a week after the stacks of agar plates had initially been placed in the incubator. The diameters of the zones of inhibition were then measured with a ruler and recorded to the nearest millimeter. After measuring the zones of inhibition, a gram-staining technique was used to illustrate the difference in cell wall structure between S. aureus and E. coli. First, a small sample of each species of bacteria was transferred with a loop from the agar plate onto two different areas on a microscope slide that had previously been washed, wiped with alcohol, and dried. Then a drop of water was added to each bacterial sample, was smeared into a thin layer, then allowed to air dry. Once the bacterial samples were dry, the slide was held at one end and was passed through a Bunsen burner flame with the smeared-bacteria samples facing up.

In this experiment, each student was randomly assigned with a different species of gram- negative bacteria. The organism that I was assigned was Unknown #16. The identity of the gram-negative bacteria was determined to be Escherichia coli. The purpose of this report is to describe the various tests that helped develop a better understanding of the unknown microorganism in terms of the physiology, morphology, motility, and antimicrobic sensitivity it is characterized with. Indole production, hydrogen sulfide, and the colony morphology on the Eosin-methylene blue (EMB) plate, were the critical results that led to the conclusion that the organism was E. coli. In the indole production test, E. coli was one of two organisms,

Introduction The purpose of this experiment was to test whether the soil from outside the Suprenant building contained the highest level of bacteria out of our three samples: soil, human lips, and a toilet seat. We determined that the soil should contain the highest level of bacteria. A teaspoon of soil can contain anywhere from 100 million to 1 billion bacteria (Ingham n.d.). This was the basis for our assumption that the soil would contain the highest levels of bacteria.