Week 01 experiments

Week 01 experiments - BIOL519 Week#1 BACKGROUND The overall...

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Week #1 1 BACKGROUND: The overall goal of the experiments this week is to remember/learn some of the fundamental microbiological techniques that we will be using this semester. Experiment 1 - Streaking for isolated colonies using toothpicks: The goal here is to adapt the methods for streaking a plate to obtain isolated bacterial colonies, to the use of toothpicks. When performing bacterial genetics, one must often streak out large numbers of colonies at a time. Using individual sterile toothpicks to perform the streaking greatly speeds this process compared with using an inoculating loop that must be flamed and cooled at each step. Instead, each time a sterile tool is needed, a new toothpick is simply taken from the beaker. (See the attached procedure for “streaking a plate with toothpicks”.) We use flat toothpicks that have been sorted so all the “fat” ends point down in a 50 ml beaker. As the sorting process is time consuming, and the toothpicks actually improve with age (they accumulate bacterial cells and agar in them, and thereby become smoother, and easier to streak with), we will routinely return the used toothpicks to a non- sterile beaker for re-autoclaving. You will first streak a bacterial strain onto a full plate using toothpicks, to become acquainted with the technique. As I mentioned earlier, bacterial geneticists often need to streak out relatively large numbers of colonies at a time. In order to save plates, we often will streak up to 8 different isolates on the same plate. It is important to become proficient at obtaining isolated colonies even when streaking in a small area of a plate (such as a 1/8th sector). We will practice this by streaking the same bacterial strain onto 6 individual sectors of a single plate - attempting to obtain isolated colonies in each sector. Experiment 2 - Patching: Another very commonly used technique in bacterial genetics is “patching”. This involves drawing a small line of each of up to 100 different bacterial isolates onto the surface of a single plate. In this way, hundreds or thousands of different isolates can be tested for a particular phenotype (such as, can they grow on a given type of media, what is their color on a particular type of indicator media, etc.) relatively quickly, and using a small number of plates. In this technique, the goal is not to obtain isolated colonies, but simply to grow a small sample of the strain in a defined location on a plate. In order to define the position of the individual isolates, a template with 25, 50 or 100 evenly sized, numbered squares is placed under the plate(s) onto which you wish to patch. If you need to test the same isolates on more than one type of media at a time (either to test multiple phenotypes, or to include a control), then the same isolate is simply patched into the same numbered square on each of the plates to be tested (i.e. - isolate #1 is patched into square 1 on each plate, isolate #2 is patched into square 2 on each plate, etc.). To make this process much
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Week 01 experiments - BIOL519 Week#1 BACKGROUND The overall...

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