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NAU BIO 205L - BIO205_Prelab5

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Kristina SimonovicSection O17 Feb 2016Exercise FiveTitle:Enumerating Bacteria, Storage Conditions, Staining & Environmental IsolateHypothesis:If all of the procedures are done aseptically, then the specimen won’t be contaminated and it will be easily identified under the microscope.Methods:Clean and sterilize the blender with QT, then rinse the blender with RO water and dry it by placing it inverted on a paper towel. Weigh 10g hamburger and put it in the blender. Add 90mL 0.75% NaCl. Blend for 10 to 20 seconds at a time, blending thoroughly to break up fat globules. Use a sterile 25mL pipet and dispense 1mL into 15mL labeled conical tube. Label the 5 culture tubes and aseptically transfer 4.5mL of 0.75% NaCl. Select a tube and record the identification letter of the tube for future reference. This represents a 1:10 dilution. Make 1:10 serial dilutions of your hamburger solution. Mix the contents of the hamburger to the 10^-2 dilution tube using a1mL pipette. Mix the 10^-2 dilution tube well and transfer 0.5mL from the 10^-2 tube to the 10^-3 tube. Make dilutions in the same manner for the rest of the tubes. Starting with the 10^-2 dilution, remove 0.1mL and place it onto the surface of the plate marked 10^-3 spread. Dip the glass hockey stick into ethanol and ignite the ethanol using a Bunsen burner. Smear the 0.1mL evenly over the surface of plate. Repeat for the other tube dilutions except for the 10^-6 tube. Next, add 0.1mL of sterile diluent to control plate. Starting with the 10^-3 dilution, place 1.0mL into the empty petri dish labeled 10^-3 pour. Retrieve the flask containing molten NA from the waterbath and pour just enough agar into the plate to cover the bottom completely. The lid of the petri dish is replaced and the 1.0mL sample and the agar are gently mixed by rotating plate in a figure 8 fashion. For a control, add 1.0mL of sterile diluent to an empty control plate and pour enough molten NA to cover the bottom of the petri dish. Once the pour plates are cool, tape all the pour plates together and all the spread plates together and incubate at room temperature. Continue with the environment isolate Safety Concerns:1. Do not pipet any sediment or fat globules found in the bottom of the blender or floating on top.2. Do not get oil on the 10 and 40 power lenses3. Make sure to label all the tubes and plates correctly4. Wear safety goggles when working with stains5. Wear lab coat at all timesRelevance:It is important to learn the aseptic technique when trying to identify microorganisms, because if the specimen is contaminated, one won’t be able to properly identify


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