Medical Microbiology

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Pure Culture Techniques

In this first lab, you will be learning some very fundamental and important techniques. As is the case with most things, shorts cuts usually get you in trouble. This is especially true in Microbiology. The techniques you will be learning tonight, if mastered correctly, will make your life and learning experience in Microbiology much easier, if you don’t pay attention and practice these techniques incorrectly, well then……? Today you will be learning the following techniques: 1. Streak plate method for colony isolation

2. Aseptic transfer techniques
First the streak plate methods. In Microbiology it is often necessary to isolate pure colonies of one bacterium from mixed cultures, or to check the purity of cultures. You can do this by “spreading” the bacteria on a nutrient growth media in a specific manner as demonstrated in the lab tonight. In this lab we will be learning two methods to accomplish this task, the quadrant streak and the T streak.

1. Quadrant streak

2. T streak
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Lab exercise:
Following the demonstration of both of these isolation techniques, it is your job to see how well you can repeat the procedure, and get isolated colonies of bacteria. A colony of bacteria is nothing more than a visible mass of bacteria that has grown on a plate as a “descendent” of one bacterium that was deposited at that spot by the streaking technique. Isolated colonies, as the name implies, are masses of bacteria that are clearly separated from one another on the plate. Bacterial colonies that have a different shape, size, or color are generally different bacteria. In this exercise you are streaking only one bacterium, so all the colonies should look pretty much the same. You will need the following: 2 tryptic soy agar (TSA) plates

Broth culture of Escherichia coli

After you have streaked your plates, they will be incubated at 37°C. The plates MUST be labeled and incubated in an inverted position. Next lab we will examine your technique!

Aseptic Transfer Technique

The term aseptic transfer refers to a set of procedures that are used to minimize the chances that a culture you are working with will become contaminated by organisms from the environment. In the microbiology lab this is a technique that you must learn, practice, and perform with the greatest of care. EVERY time that you open a culture of bacteria to perform any manipulation you must carry out this set of procedures. The procedure is not hard, but does follow a specific routine of manipulation of tubes and inoculation devices, loops or needles. One thing that will really help when you do transfers is that your work area is well organized, and not cluttered with other materials. In other words, get things set up in advance before you do transfers!

In the course of this semester in this lab you will be doing hundreds of transfers from a variety of media, both liquid and solid. The technique that will be demonstrated is exactly the same for ANY media. At first these techniques may seem a little awkward, but with practice these techniques will become second nature to you. Please remember to always follow the steps in the procedure EXACTLY, taking short cuts, or sloppy technique can really get you into trouble in this class. Here is summary of the technique: 1. Sterilize your loop until the entire thin length of the loop, not just the end, is red hot in the burner flame. 2. Allow the loop to cool for a few seconds.

3. Pick up the tube from which you want to transfer bacteria. Remove the cap, but KEEP THE CAP IN YOUR FINGERS, DO NOT PLACE THE CAP ON THE BENCH! Briefly flame the mouth of the tube, and then put your sterile loop into the culture tube to obtain a sample of bacteria. Remove the loop, re-flame the mouth of the tube, and replace the cap. Put the tube back in the rack. 4. Pick up the tube into which you want to transfer (inoculate) the sample of bacteria. Remove the cap as before,...
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