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NAU BIO 205L - BIO205_Prelab9

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Kristina SimonovicSection O23 Mar 2016Exercise NineTitle:Bacteriophage One-Step Growth Curve and Determination of Burst SizeHypothesis:If dilutions are done aseptically, then the phage growth cycle can properly be studied.Methods:Aseptically transfer exactly 9.9mL of LB broth into 12 of the sterile 19 x 150 tubes, and exactly 9.0mL of LB broth into the remaining 3 tubes. Arrange the tubes in an appropriate rack so that there are two rows of seven tubes each. In the first row, place seven tubes containing 9.9mL of LB broth. In the second row, the first three tubes will have 9.0mL of LB broth and the last four tubes 9.9mL. Label the remaining tube containing 9.9mL of LB broth DIL and place in the 37 degree C water bath. Transfer 4 mL of melted LB soft agar into the seven 13 x 100 sterile tubes. Label the seven LB plates with your group’s initials, date, and 15,25,30,35,40,45,and 50 & place them in the 37 degree C incubator. To begin the experiment, transfer the tube containing bacteriophage T4 from the ice bucket to the 37 degree Celsius water bath and allow the tube and the phage to come to 37 degrees. When everything is ready, vortex well the standing overnight culture of E. coli strain B, then transfer 2.9 mL into the 37 degree Celsius ADS tube containing the phage. Mix the cells and phage gently by finger vortexing. This is time zero. Your timekeepershould note the exact clock time of transfer in the table. While waiting for the five minutes required for absorption to elapse, the timekeeper should record the clock times in the table belowindicating when each sample should be removed for dilution and plating. After the five minutes, gently mix the ADS tube and aseptically transfer 0.1 mL to the DIL tube. Gently mix the DIL tube and place it back in the 37 degree Celsius water bath until the next time point (10 minutes later). You will remove all of the remaining samples from the DIL tube. The timekeeper will inform you just before you need to take the 15 minute sample. At this time, you should gently mix the DIL tube by finger vortexing and transfer 0.1mL from the DIL tube into the first tube in the front row of your rack. After the transfer, gently mix the first row dilution tube and then transfer 1.0 mL from it into the tube behind it in the second row. Get a tube of soft agar from the 47 degree Celsius water bath and add 0.3 mL of E. coli B lawn cells. Gently mix the second row dilution tube and transfer 0.1 mL from it into the LB soft agar and E. coli and immediately pour onto the plate labeled “15” and spread evenly. Discard the first and second row dilution tubes and the tube used for the soft agar into the appropriate container. Make sure you keep the DIL tube in the 37 degree Celsius water bath between sampling points. Repeat this procedure for the 25 and 30 minute time points. For the remaining time points (35, 40, 45, and 50 minutes), there is only one difference from the protocol in step 5: instead of transferring 1.0 mL from the front row tube, you transfer 0.1 mL. At this point, you have diluted the staring phage concentration (ADS) by 10^-6. After the soft agar overlays have solidified, tape the 7 plates together and incubate at room temperature upside down for 24-48 hour. During the next lab period, you willcount the number of plaques (clear zones in the confluent growth of the bacterial lawn). Record your results and complete the worksheet that will be provided.Safety Concerns:1. Use aseptic technique to insure the sample is pure2. Make sure to prepare all steps before beginning the lab3. Samples will be taken at timed intervals4. Properly label all tubes for accurate results Relevance:Viruses infect animals, plants, and even bacteria. A fundamental part of microbiology is studyingthe progress of the phage growth cycle by taking samples of the infected culture over time and assaying the number of


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