The purpose of this experiment is to determine the effects of the major component of respiratory burst on pathogenic Enterohemorrhagic Escherichia coli. Respiratory burst (or oxidative burst) is the rapid release of the reactive oxygen species (ROS), superoxide anion (O−2) and hydrogen peroxide (H2O2), from different cell types. Respiratory burst is used by immune cells like macrophages and neutrophils, to degrade internalized pathogens through lipid peroxidation and DNA destruction.
Escherichia coli is a Gram-negative bacterium that is a normal component of the human intestinal flora and is ordinarily not a dangerous organism. The K12 and related strains of E. coli used in molecular cloning have been modified so that they are able to survive in culture only under very specific conditions and are unable to survive at all in the human gut. There are, however, some types of pathogenic strains that can cause serious harm to human. The diverse disease types caused by E. coli have resulted in their classification into distinct pathovars such as enterohaemmorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and enteroinvasive E. coli (EIEC).
As mentioned in the first paragraph, endogenous reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anion radical, and hydroxyl radicals are generated by immune cells and are responsible for damages on nucleic acids (RNA and DNA), as well as proteins and lipids, leading to microbial cell death. Defense mechanisms against the damaging effects of oxidative stress involve both enzymatic components, such as catalases and superoxide dismutases (SOD), and nonenzymatic components, such as glutathione-dependent reduction systems. It has been previously thought that E. coli pathovars have the ability to resist destruction by repiratory burst by producing enzymes that break down catalases and superoxide dismutases (SOD).
In this experiment, normal E. coli (non-pathogenic species isolated from the human gut), K12 E. coli, and pathogenic E. coli were exposed to H2O2 (hydrogen peroxide) at a post 1.0 absorbance at 600 nm (A600). Optical Density (OD) measurements of microbial and cell growth are one of the most common methods used in a microbiology lab. Some of the main applications are the determination of the optimal time at which to harvest, the determination of the optimal time to induce a culture when running a protein expression protocol or the monitoring of cloning procedures. The growth of cells, bacteria or yeast (cell density, bacterial growth, yeast growth) in liquid culture media is commonly controlled by measuring the optical density at 600 nm (OD600).
OD600 measurements are typically used to determine the stage of growth of a bacterial culture, these measurements help ensure that cells are harvested at an optimum point that corresponds to an appropriate density of live cells. Growth of bacterial cells typically progresses through a series of consecutive phases including lag, log, stationary, and decline.
In general, cells should be harvested towards the end of the log phase, using the optical density of the samples to determine when this point has been reached. Cells are routinely grown until the absorbance at 600 nm (known as OD600) reaches approximately 0.4 prior to induction or harvesting.
At the indicated times (indicated by a letter A L), 100µL (0.1mL) of the bacterial broth culture was taken out and serially diluted 8 times. The 10-6, 10-7 and 10-8 plates were plated (dilution factors are indicated on the Excel sheet). On each of the plates, 100µL (0.1mL) was plated (plated volume). Each time point extraction was plated onto 3 plates (3 technical replicates) to account for pipetting errors and growth challenges (to run statistics).
Go to the Growth Curves section. Plot the graphs of all three types of E. coli. Does the addition of hydrogen peroxide affect any of the growth curves? If so, please describe the effect? (Please include these graphs in your report)
Using the data in the Cell Viability section, calculate the average and standard deviation of the CFU/mL of each time point. (Hint: remember to put numbers into scientific notation and don’t forget the equation (CFU x Dilution Factor)/(Volume Plated)). Be sure to keep an eye on the dilution factor and remember you should have 1 average per letter time point (AL). Use these averages to graph column graphs of the data and include these graphs (with error bars representing standard deviation) in your report).
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