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Transfer and Isolation Techniques, Microbes in the Environment

Objectives After completing this lab, you should be able to: 1. Transfer bacteria aseptically between tubes of growth media. 2. Aseptically perform a streak plate resulting in isolated colonies. 3. Properly prepare a pour plate. 4. Determine that microbes are virtually everywhere on environmental surfaces Tube-to-Tube Transfers It is often necessary to take a sample of bacteria from The type of microbe that will most often be used in this laboratory will be bacteria (singular _ bacterium). one tube and place it into another. The procedure is known as an inoculation. The process requires care Bacteria are usually grown on material known as a and growth medium (plural _ media). The chemical or precision, and it is performed by following nutritional established composition of media is extremely varied and will be covered later in this course. Media are utilized procedures or protocols generally considered aseptic technique. Aseptic technique will be an integral part in several different forms. The most common are of liquid every laboratory session in this course and will be broth, agar slants, agar deeps, and agar plates.* used Agar is a chemical derived from seaweed that solidifies into daily when you ultimately become a member of the medical or allied health professions. a jellylike semisolid. When nutrients and other Use of aseptic technique in the transfer of bacteria growth from one tube to another: factors are added, many different kinds of bacteria 1. Prevents the microbe in the tube from can be grown on it. When a melted agar solution is poured contaminating the work area or the person performing the transfer. in a tube, tilted, and allowed to solidify, it is called a 2. Prevents microbes in the work area or those on the slant. When melted agar is poured into a tube and person performing the transfer from contaminating allowed or mixing with the microbe being transferred. to solidify without slanting, it is called a deep. Basic Aseptic Technique Procedures When placed in a flat dish or plate (Petri dish), it is Flaming the Inoculator. An inoculator is a thin wire called an agar plate. attached to a handle used to aseptically transfer All media used in this class will be sterilized in a bacteria device called an autoclave, which uses steam under pressure (the inoculum) into various types of growth media. If the end is twisted into a loop, the device is called an to destroy all known infectious agents. If time inoculating loop. If it simply comes to a point, it is permits, called your laboratory instructor will demonstrate this an inoculating needle or stab. You will be using the device inoculatingloop for most procedures in this course. The loop is heated before and after all procedures in order to destroy any contaminating microbes present


on the loop itself. Figure 2.1a demonstrates the proper procedure for heating the inoculator in a burner. Grasp the inoculator (loop or needle) with your dominant hand, as you would a pen or pencil. Then heat the loop and the wire to redness by holding it at approximately a 60degree angle just outside the blue cone of the Bunsen burner flame. After flaming, the heated loop and wire should not be allowed to touch the countertop or any rubber tubing. Note: If you are sharing a burner with another student, you must take care not to heat both inoculators at the same time. Otherwise someone may get singed. Many laboratories have changed their methods of heating the inoculator by using a device called an incinerator (Fig. 2.1b). An incinerator is a tube surrounded by a wire grid imbedded in a ceramic cylinder called the heater element. Heat is generated by an electric current delivered through the heater element. All standard transfer techniques can be performed by using this device. Most incinerators require a warm-up time of five minutes to reach optimum temperature, so you should plan ahead if you use an incinerator. However, once in operation, it can remain on for the entire laboratory session. All microbes will be destroyed if the inoculator remains in the incinerator for five seconds. Make sure you place the tip of the inoculator far back into the ceramic tube to avoid potential splatter of inoculum. Holding Transfer Tubes. You hold the tubes used to transfer bacteria in your nondominant hand. (If you are right-handed, use your left hand; if you are lefthanded, use your right hand; and if you are ambidextrous, its up to you.) Hold both tubes together in your hand so that they are parallel to each other and touching. If either or both tubes have a screw cap, loosen the cap(s) to the point where they will lift right off. If either or both tubes have pop-off caps, they can usually be removed