After the successful run of the serial dilution method for the E.Coli B colonies, the same serial dilution method was used (beginning with the start concentration of 50,000 cells per mL) and combined with the two phage cultures T2 (Pic 1) and T4(pic2). Similar results to the control serial dilution can be seen; however, when combined with the phages, the dilutions of 50, 5, and .5 cells per mL have noticeable less colonies, almost none. These are promising results. The experiment will be run again for confirmability and the addition of more time points to gather more data on how the phages kill. Data was entered into the spreadsheet for further data analysis. Also, time will be taken to gain a deeper understanding of Michaelis-Menten Kinetics to enrich the understanding of the project, our results, and conclusion.
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For week 16, another dilution series was run to get baseline data for verify the validity of the dilution method being used to plate the E.Coli B colonies. Using the flamed bent glass method (pic 1) 12 plates were inoculated with a series of increasingly dilute E.Coli broth cultures using the serial dilution method where a 1:9 ratio is used of infected:clean broth in succession to reach the desired dilution range. To get a good starting point, a cytometer slide (pic 3) was used to count cells in the original culture and when only one cell of E.Coli could be seen the broth had a concentration of 50,000 cells per mL. Results analyzed after sufficient days of growth showed success of the dilution process, all colony counts were entered into the spreadsheet for later data analysis.
For week 15, it was decided that a new set of all strains needed to be plated. After re-plating the specimens, the newly-filtered stock of bacteriophages, both strain T4 and T2, were tested for contamination. The phages were also tested for viability; a small sample of the phage and E.Coli broth was taken out of one of the sterile filtration setups before it was filtered through, it was plated against some broth of pure E.Coli (picture 1) and as can be seen in the picture, no E.Coli grew, indicating a positive result. To get a set of baseline data for the core experimentation, a gradient of different ratios of phage:E.Coli were grown in a series of snap tubes and plated out to see how many colonies grow based on the ratio of phage to culture (photos 2-6). These results will be gathered and tweaked to get a solid set of baseline data for the core of the experimentation.
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