Biol 2281, Summer 2013 E3: Microbial Techniques Procedure/Report Handout
Experiment 3: Microbial Techniques
Objectives: By the end of this lab, you will be able to: 1. Understand and practice aseptic techniques in handling microorganisms. 2. Learn simple media preparation procedures 3. Streak plates and spread plates to obtain single colonies. 4. Understand how serial dilutions are made and how cell density can be determined through standard plate count. 5. Learn to use micropipettes and other types of pipets with pipet pumps. 6. Understand the concept of replica plating. Note: At the end of the lab, your agar plates will be incubated at 30˚C for approximately 48 hours and then placed in a refrigerator. You will complete your lab report after analyzing your plates during next week’s lab session. A. Sterile and Disinfection Techniques 1) Sterilizing with moist heat: Moist heat provided by an autoclave or pressure cooker at a pressure of 15 psi (at 121OC) is an efficient way to sterilize most materials. 2) Sterilizing with dry Heat: Dry materials such as glass and metal may be sterilized in an oven, at 160O C for two hours. 3) Sterilizing by flaming: The flame from a gas burner effectively sterilizes small glass or metal objects, such as inoculating loops. Flame the metal inoculating loop until it is red hot. In order to prevent killing of the microorganisms, you must remember to always cool the flamed inoculating loop by letting it briefly touch sterilized liquid or solid media. To sterilize metal or glass spreaders, dip the spreader in alcohol and then pass the spreader through the flame to ignite the alcohol. 4) Disinfecting the bench with 70 % alcohol: Remove mold-laden dust, the most common source of contamination, by wiping the bench with 70% alcohol. If your skin is not particularly sensitive to alcohol, wipe your hands with a small amount of alcohol, too. 5) Principles to eliminate or minimize airborne contamination or contamination caused by human skin or secretions: • Keep containers for growing microorganisms, such as Petri-dishes, culturing tubes, covered with lid or cap as much as possible. • Don't touch anything that have been sterilized and will be in direct contact with microorganisms, such as the micropipette tips. • Wipe down the surface around the experiment with alcohol and minimize air turbulence. • Avoid talking, singing, whistling, coughing, or sneezing in the direction of things that should be sterile. Long hair, if not tied back, may be a source of contamination. B. Growth Media There are different types of media for growing different types of cells. Bacteria and yeast can be grown on solid media or in liquid media. LB (Luria Broth) media which contains tryptone, yeast extract and NaCl is the most common liquid media for growing bacteria. Solid media have the same chemical ingredients as the liquid media except that they contain an additional solidifying 1
Biol 2281, Summer 2013 E3: Microbial Techniques Procedure/Report Handout agent, typically agar. The concentration of agar for conventional agar plates is 2% (2 grams of agar per 100 ml of final volume). In making the media, the container should not be filled to more than 2/3 of its capacity. For example, you can prepare no more than 600 milliliters of media in a 1000 milliliter flask. If you fill containers too full they may boil over during the sterilization process. It takes approximately 25 milliliters of media to pour one standard 100 x 15 mm plate (Petri dish). Two experiments in BIOL2281 will use one or more of the following types of media: 1) YED medium (Yeast Extract + Dextrose) will be our standard growth medium for yeast. Red adenine-requiring mutants grow well on this medium and develop the characteristic red pigment. 2) YEAD medium (Yeast Extract + Adenine + Dextrose) contains the same ingredients as YED but has excess adenine added. Red adenine-requiring yeast mutants grow well on this medium. 3) MV medium (Minimal plus...
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