Lab 11 Background

Phage Killing Assay

Phage killing assay is a direct way to assess the virulence of phage by infecting bacteria with the phage of interest. In our experiment, we will treat identical bacteria with either the WT or the mutant T7 phage, and allow the phages to kill the bacteria. We will assess the killing efficiency of the phages by measuring the number of bacteria at different time points. The more virulent the phage, the quicker the killing, and the smaller the bacteria number measured.

The Importance of Controls in Scientific Experiments

The use of proper controls is arguably the most important thing when conducting scientific experiments. A control is used in an experiment to minimize the effect of variables that are not an interest in the study. In other words, control is used as a benchmark for comparison against which experimental results are measured. Without proper controls, no association or causal relationship between the testing variable and the experimental result can be confidently determined.

To identify proper controls, one needs to consider all variables in the experiments. For example, in this lab, the goal is to test the virulence of the WT and mutant T7 bacteriophage. To do so, it is important to use the same number of bacteria cells that are genetically identical and the same number of T7 bacteriophage particles. If different number of bacteria or viral particles are used between the two conditions (WT and mutant) in this experiment, a conclusion cannot be made on the virulence of the phages since these other variables may contribute to any difference in the experimental result. To achieve the former, a single strain of bacteria (10G) will be cultured in a tube and then aliquoted to be used in the experiment as the host for both the WT and the mutant phages. To achieve the latter, the same number of T7 particles will be used in the experiment.

Additionally, it’s important to include another aliquot of the bacterial cells without any phages as the “negative control” (without adding an experimental variable, in this case, the T7 phage). This control is used as a benchmark for normal bacterial growth. Without this control, a conclusion cannot be made on the virulence of the WT T7 phage, which renders the difference between the WT and mutant phages obscure. This control is also used to show that the experimental condition/apparatus works as planned. Since the WT T7 phage infects and kills the 10G E. coli bacteria, if the number of bacterial cells is not reduced in the WT T7-treated sample compared to the non-treated sample, it is suggestive that the experimental setting is wrong and troubleshooting is in order.

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