For this experiment we will run the autoclave for only 5 minutes. We will test 3 different volumes of deionized water to see if there is a similar effectiveness at killing bacterial endospores using the BT Sure spore test ampules. Ampules will be suspended in each of the the volumes of liquid using a string so that the spores are close to the “center” of the liquid in the flask. NOTE: See BT Sure Bacillus spore test ampule product sheet in folder with this Protocol sheet. Experimental Protocol 1. Set up 3 Erlenmeyer flasks with the following volumes of deionized water: a. 2-liter flask with 2 liters of water. b. 500ml flask with 500ml of water. c. 250ml flask with 200ml of water. 2. Suspend a BT Sure ampule in each flask using a string so that each ampule is in the middle of the liquid rather than touching the flasks surfaces. 3. Set up appropriate postitive and negative control samples. 4. Place the flasks in the autoclave set for liquids and a 5-minute run time. (Other conditions will be standard conditions of 121oC and 15 lbs/in2. 5. Following the completion of the run (when the door can be opened) remove the flasks immediately. 6. Using forceps remove the ampules from the flasks and place at room temperature for 10 minutes to let them cool down. (Use caution here. Ampules may burst if they cool too fast. Place them in a protected area to prevent injury.) 7. Place the ampules in room temperature water for another 5 minutes. 8. Break the inner medium tube in the ampules and mix the contents with the spore-containing paper. 9. Place all ampules into an incubator and carry out the incubation of the spores according to the manufacturers instructions. 10. At the appropriate time assess the ampules for bacterial growth. Complete the table below. Ampule Observation negative control positive control 2-liter water 500 ml water 200 ml water Name: Cell Culture Techniques Spring 2021 Experiment #2 – Effect of Liquid Volume on the Effectiveness of Autoclaving for Short Times Writing Assignment – Pre-Lab (10 points) Assigned: 2/16/21 Due Date: 2/19/21 by 9:00am – (NOTE: For this assignment you will not be allowed to hand this assignment in late or you will lose 1/2 credit.) Answer the following questions. Use complete sentences and be thorough in your responses. Use blue italics for your responses. 1. What is the scientific name for the organism that is being used in this experiment? 2. State a hypothesis for this experiment. Be sure you include an educated guess for the outcome of the experiment. 3. What is the independent variable in this experiment? 4. What is the dependent variable in this experiment? 5. Describe what the negative control should be for this experiment and explain why it is being included in this experiment. 6. Describe what the positive control should be for this experiment and explain why it is being included in this experiment. 7. Explain what a positive and a negative result will look like and explain what each result means. 8. Fill out the following table for what you expect to observe for each ampule in this experiment using the table below. Ampule Observation (expected) negative control positive control 2-liter water 500 ml water 200 ml water Requirements: long enough they dont need to be super in depth ‘
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