Aseptic Technique and Streak Plates
The purpose of this exercise is to learn aseptic technique procedures and there importance and also to learn to isolate colonies using the streak plate technique. Introduction
Bacteria are inoculated (introduced) and cultured (grown) in the laboratory for test studies to determine their morphology (the shape, size, arrangement, and internal structures) and pathology (ability to cause disease). Inoculation has to be performed without adding other microbes or contaminants. Aseptic (sterile) technique is the process of growing pure (uncontaminated) cultures, and is essential for proper characterization of a bacterium. Aseptic technique includes the use of sterile media and equipment. The sterility of these tools is maintained by proper handling procedure, which prevents the introduction of contamination. The basics of aseptic technique are: Autoclave or steam sterilization of all equipment and media. Flame sterilization of wire loops and needles which are used to transfer bacteria from one growth condition to another. Flaming the mouth of test tubes and other containers to establish hot air convection out of the container, to reduce the chance of microbes entering the tube by air flow. Mixtures of organisms, as found in nature, will have characteristics of the group and thus identification of a single member of the group is not possible. In order to determine the cause of a disease the microbe must be separated from the mixture and cultured as a pure culture. The standard method of obtaining a pure culture is the streak-plate method on agar. The streak-plate method is an example of a solid dilution technique. Bacteria are obtained from a mixed culture with a sterile loop or wire and diluted within three sectors on a petri dish containing appropriate growth medium. After incubation, as a result of this dilution technique, individual colonies of microbes can be obtained. Each individual colony represents the descendents of a single cell, about 1 million (106) bacteria are necessary to be visible to the naked eye. By inoculating the individual colony with a sterile loop onto on a fresh agar surface, a pure culture may be obtained. Today’s laboratory exercise will give you the guidelines for performing aseptic technique when working with bacteria, not only for keeping the cultures pure, but for safety reasons. You will use this technique for the remainder of the laboratory exercises.
Per group of four students:
1ml broth culture of Escherichia coli
Plate culture of E. coli
Striker – for lighting the Bunsen burner
2- Culture tubes of Brain Heart Infusion (BHI) Media
2- Trypticase Soy Agar (TSA) plates
1 – Wire Inoculating Loop
Note before you begin: Bunsen burner should be adjusted so that the flame is blue. You will see a dark blue area in the center of the flame and a light blue area at the top of the flame. The light blue area is the hottest area of the flame. Broth to Broth Transfer
1. Obtain two tubes of BHI broth and label them with your initials, date, and either “EC” for Escherichia coli or “C” for control. 2. Grip the wire loop the same way you would hold a pencil. Hold the wire loop at an angle so that you do not burn your hand, see figures 1-3. Flame the wire loop until it is red hot. Allow the loop to cool for a few seconds. Do not blow on the loop or wave it around to cool it. 3. Pick up the tube of E. coli with your other hand. Remove the test tube cap by curling the little finger and fourth finger of your right hand (the hand holding the loop) around the cap to hold it. See figure 4.
* Figures 4-7. Remove caps from liquid specimens and replace the caps of the test tubes with the same hand that holds the loop. The caps must be held during the entire procedure, as shown below in Figures 4-6, and not placed on the desktop or contamination may result. Flame the...
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