Unknown #2 Assignment (20 points total) 1. You are given a mixed broth culture of Staphylococcus aureus and Escherichia coli. You are instructed to isolate and purify each bacterial species. How are you going to do it? Your goal is to have a plate of pure S. aureus with isolated colonies and a plate of pure E. coli with isolated colonies. Explain in detail how you are going to perform the exercise. You can use any media you like such as nutrient agar plate, trypticase soy broth (TSB), MSA, EMB, MacConkey agar, etc. You can also use any lab equipment that we have used in the lab. Pay attention to the incubation temperature, incubation time and oxygen requirement of the organisms that you are culturing. When trying to explain to me how you are going to perform the exercise, imagine that you are giving instructions to your son or daughter. Handwritten work will not be accepted. No copying, plagiarizing or cheating. No collaborating. Work independently. 2. You are given a mixed broth culture of Pseudomonas fluorescence and Clostridium sporogenes. You are instructed to isolate and purify each bacterial species. How are you going to do it? Your goal is to have a plate of pure P. fluorescence with isolated colonies and a plate of pure C. sporogenes with isolated colonies. Explain in detail how you are going to perform the exercise. You can use any media you like such as nutrient agar plate, trypticase soy broth (TSB), MSA, EMB, MacConkey agar, etc. You can also use any lab equipment that we have used in the lab. Pay attention to the incubation temperature, incubation time and oxygen requirement of the organisms that you are culturing. When trying to explain to me how you are going to perform the exercise, imagine that you are giving instructions to your son or daughter. Handwritten work will not be accepted. No copying, plagiarizing or cheating. No collaborating. Work independently. (Name) Bio. 152 Dr. Perez 15 April 2020 Unknown #2 Assignment 1. Isolating Staphylococcus aureus and Escherichia coli: In order to isolate and purify each bacterial species from the mixed broth culture of Staphylococcus aureus and Escherichia coli, we have to use the streak-plate isolation method and this can be done through these steps: a. Take a plated agar medium and label its base with your name, date, and names of the broth mixture. b. Incinerate the loop from base to tip to sterilize it and then take the sample of the mixed broth culture by shaking the broth and flaming the mouth of the tube and then entering the loop until the base and obtain the sample. c. Keep the sterile agar plate on the table, lift the lid gently and then start with the edge of the plate, lightly drag the loop back and forth with the loop touching the agar, but not scraping it. d. Take off the loop and sterilize it again as before, in the meanwhile replace the lid to prevent contamination. e. Rotate the plate a little less than 90°. With a sterilized cool loop, start another streak from one end of the first streak and intersect the first on two or three times only. f. Sterilize the loop again and repeat with the third streak starting with the second one. And the same thing for the forth streak. g. Since both Staphylococcus aureus and Escherichia coli are mesophiles, their optimum temperature is 37°C, so we can incubate the plate in 37°C for 24 to 48 hours. h. After this incubation period, we will see the results of having different colony morphology, E. coli will be white and opaque colonies while S. aureus will be shiny yellow, round colonies. However, we can use another media methods the enhance the isolation by inhibiting growth of some organisms and promote the growth of other organisms such as Mannitol Salt Agar (MSA), which contains high salt concentration that will selectively allow salt-tolerant organisms like S. aureus to grow and inhibit E. coli. a. With this procedure we need to follow safety rules and wear lab coat, goggles, and gloves for protection. b. Grab one MSA plate and label its base with your name, date, and names of the broth mixture. c. As performed earlier with inoculation, Incinerate the loop from base to tip to sterilize it and then take the sample of the mixed broth culture by shaking the broth and flaming the mouth of the tube and then entering the loop until the base and obtain the sample. d. Spot inoculate the sectors on the MSA plate with the mixed broth culture. e. Invert and incubate the late at 37°C for 24 to 48 hours. f. Save and dispose the original culture is directed by your instructor. g. After the incubation period, we will see the results of having bright yellow growth or halo with means that there are organism produces acid from mannitol fermentation and obviously the presumptive ID would be Staphylococcus aureus. In order to obtain E. coli culture, we can use another type of media which is Eosin Methylene Blue Agar (EMB). This selective and differential medium inhibits growth of most Grampositive organisms (like S. aureus) and promotes the growth of Gram negative organisms (like E. coli). a. Follow safety rules and wear lab coat, goggles, and gloves for protection. b. As performed earlier with inoculation, Incinerate the loop from base to tip to sterilize it and then take the sample of the mixed broth culture by shaking the broth and flaming the mouth of the tube and then entering the loop until the base and obtain the sample. c. Grab one EMB plate and label its base with your name, date, and names of the broth mixture. d. Spot inoculate the sectors on the MSA plate with the mixed broth culture. e. Invert and incubate the late at 37°C for 24 to 48 hours. f. Save and dispose the original culture is directed by your instructor. g. After the incubation period, we should see dark growth (purple to black, with green metallic sheen) which is indicating of E. coli growth because it is gram negative and a strong lactose fermenter. Finally, in order to get isolated and pure culture of each Staphylococcus aureus and Escherichia coli from MSA media and EMB media we can transfer from a plate culture to a sterile agar plate by following these steps: a. Label the base of the sterile agar plate with your name, date, medium, and the organism you are inoculating with. b. Flame the loop from base to tip, lift the lid of the petri dish and use it as a shield from airborne contamination. c. Gently touch with a cool loop to the center of an isolated colony on the agar surface and collect the smallest amount you can see (Staphylococcus aureus from MSA plate and Escherichia coli from EMB plate). d. Remove the loop and replace the lid. e. Now we have pure and isolated culture of each Staphylococcus aureus and Escherichia coli. 2. Isolating Pseudomonas fluorescence and Clostridium sporogenes. One characteristic to differentiate between these two bacterial species is that Pseudomonas fluorescence is strict aerobic and Clostridium sporogenes is strict anaerobic; therefore, we can isolate them using Anaerobic Jar method. An indicator strip is used with the anaerobic systems, the strip is soaked with methylene blue or resazurin dye that is blue or pink when oxidized and colorless when reduced. a. Wear lab coat, gloves, and goggles b. Obtain two nutrient agar plates. Label the bottom of each plate with your name, date, and mixture culture organisms. c. Incinerate the loop from base to tip to sterilize it and then take the sample of the mixed broth culture by shaking the broth and flaming the mouth of the tube and then entering the loop until the base and obtain the sample. d. Perform quadrant streaking: Keep the sterile agar plate on the table, lift the lid gently and then start with the edge of the plate, lightly drag the loop back and forth with the loop touching the agar, but not scraping it. Take off the loop and sterilize it again as before, in the meanwhile replace the lid to prevent contamination. Rotate the plate a little less than 90°. With a sterilized cool loop, start another streak from one end of the first streak and intersect the first on two or three times only. Sterilize the loop again and repeat with the third streak starting with the second one. And the same thing for the forth streak. e. Place one plate in the anaerobic jar in an inverted position. f. If using BD system: Stick the methylene blue strip on the wall of the jar. Open the packet and place it in the jar with the label facing inward, immediately close the jar. Examine for condensation within about 30 minutes. g. Place the second (aerobic) plate and the anaerobic jar (inverted) in 37°C for 24 to 48 hours. h. Save and dispose the original culture is directed by your instructor. i. After the incubation period, we should see the results as dense growth of Pseudomonas fluorescence in the aerobic plate because it is strict aerobic and also dense growth of Clostridium sporogenes in the anaerobic jar as it is strict anaerobe. From this experiment we can isolate these two bacterial species and we can transfer them from a plate culture to a sterile agar plate by following these steps: a. Label the base of the sterile agar plate with your name, date, medium, and the organism you are inoculating with. b. Flame the loop from base to tip, lift the lid of the petri dish and use it as a shield from airborne contamination. c. Gently touch with a cool loop to the center of an isolated colony on the agar surface and collect the smallest amount you can see (Pseudomonas fluorescence from the aerobic plate and Clostridium sporogenes from anaerobic jar). d. Remove the loop and replace the lid. e. Now we have pure and isolated culture of each Pseudomonas fluorescence and Clostridium sporogenes 
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