Microbiology Task 2

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Jan 9, 2024

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Rabeca Plummer Clinical Microbiology Laboratory C453 4/18/2021 Task 2

Introduction In this series of experiments, we attempt to categorize and identify different traits of 3 individual bacteria: Escherichia Coli, Staphylococcus Epidermidis, and Saccharomyces Cerevisiae, using an aseptic technique. The aseptic technique is defined a “ Aseptic techniques refer to any method used to sterilize and maintain the sterility of an object or location, such as an operating theatre or laboratory” (Greenwood, 2019). This ensures the integrity of the experiment and the results. Failure to use an aseptic technique can skew results and lead to false conclusions. Materials -Large cooking pot -stove -tap water -test tube rack -paper towels -isopropyl alcohol -oven mitt -marker -safety gloves -coffee mug -test tube clamp -nutrient agar test tubes -petri dishes -Escherichia Coli tablet in vial -face mask with ear loops -inoculation loops -nutrient agar -18ml tubes -nutrient broth – 5 ml tubes - safety goggles -small, graduated pipet – 5ml -tea candles

-thermometer -yeast packet -bucket -disposable cups -Lighter or match -microscope slides -Barritt’s A Reagent -Barritt’s B Reagent -Methyl red Reagent -MR-VP broth -Scissors -Fructose powder vial – 0.2g -Glucose powder -0.2g -Mannitol powder -0.2g -Durham test tubes -Phenol red broth vials – 9mL -Motility Test agar 0.4% -8mL tubes -paperclips Method Method 1: Pouring Agar Plates Step 1: Gather all the required materials Step 2: Label the bottom of 6 petri dishes NA for nutrient agar, do not open the petri dishes Step 3: Place the test tube rack and agar tubes in cooking pot and add water until the level is higher than the agar level. Step 4: Slightly loosen the tops of the agar tubes to allow air to release. Turn on the stove to bring water to a boil. Step 5: Monitor agar until it is liquified. When the agar is melted, remove the tubes from the water with an oven mitt and place in a coffee mug filled with hot water.

Step 6: Pour approximately one tablespoon of alcohol onto your work surface and use a paper towel to spread the alcohol and sterilize the area. Step 7: Open one petri dish onto your work surface, open and pour one half of a test tube of your nutrient agar into the corresponding petri dish. Repeat until all agars have been poured. Step 8: Allow agar to cool, untouched in the petri dishes until solidified. Plates should remain inverted until inoculated. When agar is cooled and solid, it is ready to be used in an experiment. Step 9: Wash your hands thoroughly with soap and water. Method 2: Culturing Microbes in Broth Step 1: Gather supplies. Step 2: Wash hands thoroughly with soap and water. Step 3: Put on PPE (gloves, goggles, apron, and face mask) Step 4: Prepare the work surface by wiping it down with 1:9 bleach water solution. Step 5: Pour a bucket of bleach for item disposal. Step 6: Fill a disposable cup with alcohol and place it on your work surface. Step 7: Place the pipet in the alcohol and squeeze some alcohol into the stem. Leave the pipet in the cup with the alcohol in the stem. Step 8: Place the tea candle on the work surface and light the wick. Step 9: Use a marker to label a nutrient broth tube Escherichia Coli and place it on the work surface. Step 10: Expel the alcohol from the pipet and shake it dry. Keep the pipet in your hand and don’t allow it to touch anything, as it is now sterile. Step 11: Remove the top from the labelled nutrient broth and pass the rim through the flame of the candle to sterilize it. Step 12: Place the vial upright on the work surface and repeat with the e coli culture vial. Step 13: Pipet 0.25mL of nutrient broth into the e coli culture vial, being careful not to touch the sterilized rim on either vial with your gloves or the pipet. Step 14: Replace the lid on the culture vial and shake until the tablet dissolves. Step 15: Pipet the dissolved tablet and nutrient broth solution into the nutrient broth vial, being careful not to touch the sterilized rim with the pipet or your gloves. Step 16: Hold the rim of the nutrient broth vial in the flame again to sterilize it and screw the cap back on. Set the broth aside. Step 17: Place the pipet in the alcohol cup, squeeze alcohol into the stem, and leave the pipet in the cup.

Step 18: Repeat steps 9-17 with the Staphylococcus Epidermidis culture vial using a new broth tube labelled Staphylococcus Epidermidis. Step 19: Open the yeast packet and place ½ teaspoon of the powdered contents into an empty disposable cup. Step 20: Add ¼ cup warm water to the yeast and swirl until dissolved. Step 21: Allow the cup to sit for 10 minutes until it begins to froth. Step 22: Label a new broth tube Saccharomyces cerevisiae. Repeat steps 10-11 with the Saccharomyces cerevisiae broth tube. Step 23: Pipet 0.25mL of the yeast solution into the nutrient broth tube, being careful not to touch the rim of the tube with the pipet or your gloves. Step 25: Hold the rim of the nutrient broth vial in the flame again to sterilize it and screw the cap back on. Set the broth aside. Step 26: Extinguish the candle. Step 27: Sterilize the pipet in the alcohol and store with the candle, goggles, mask, and apron for the rest of the experiments. Step 28: Pour the remaining alcohol in the sink, dispose of the cup. Step 29: Place the two empty culture vials and the cup of yeast into the bucket of bleach. Remove the items after 3 minutes and dispose of them in the garbage. Pour bleach down the sink. Step 30: Wipe the work surface down with 1:9 bleach water solution. Step 31: Identify a location in your home where the broth tubes can incubate, upright and untouched, for approximately 48 hours. The location should be room temperature (21°C-25°C), away from heating or air-conditioning vents, out of direct sunlight, and secure from children and pets. An empty cabinet works well. If a countertop is used, place cultures in a box, such as your empty HOL box. Use the thermometer to determine whether the location meets the requirement of 21°C-25°C, if not, identify a new location. Step 32: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water. Step 33: Allow the tubes to incubate for 48 hours. Check for grown by holding the tubes near a light source. (Developed cultures will be either cloudy or have flocculent growth at the bottom of the tube.) Step 34: If the cultures show no signs of growth after 48 hours, incubate for an additional 24 hours. Method 3: Isolating Individual Colonies Step 1: Gather the developed cultures of Escherichia Coli, saccharomyces cerevisiae, and e. epidermidis, 3 agar plates and the rest of the required materials from the above list. Step 2: Wash your hands thoroughly with soap and water.

Step 3: Put on your PPE; gloves, goggles, apron, and face mask. Step 4: Wipe down your work surface with 1:9 bleach water solution. Pour a bucket of bleach for item disposal. Step 5: Fill a small disposable cup halfway with alcohol and place the 3 inoculation loops in the cup, loop side down. Step 6: Label the agar side of the plate with the name of the microbe it will contain. Step 7: Light the candle and place it on the work surface. Step 8: Place the agar plate next to the broth tube and remove the plate lid. Step 9: Remove an inoculation loop from the alcohol and shake it dry. Step 10: While holding the inoculation loop in your hand away from touching anything, uncap the Escherichia Coli broth and pass the rim through the flame to sterilize it. Step 11: Submerge the sterile loop into the broth being careful not to touch the rim with the loop or your gloves. Step 12: Immediately transfer the broth adhering to the loop into one quadrant of the agar plate using a zig zag motion. Replace the lid of the agar plate. Step 13: Put the inoculation loop in the alcohol cup. Step 14: Flame the rim of the broth and close the cap. Step 15: Remove the inoculation loop from the alcohol and shake to dry. Step 16: Open the lid of the agar plate and touch the loop to the edge of the inoculated quadrant to transfer the microbes to the next quadrant. Replace the lid of the agar plate. Step 17: Place the inoculation loop in the cup of alcohol. Step 18: After 20 seconds, remove the inoculation loop from the alcohol and shake to dry. Step 19: Repeat steps 16 and 17 until all 4 quadrants have been streaked. Step 20: Place the used inoculation loop in the bucket of bleach for 30 minutes and then dispose of it in the garbage. Step 21: Repeat steps 8-21 for the Saccharomyces cerevisiae and Staphylococcus Epidermidis samples. Step 22: Extinguish the candle and store for future use. Step 23: Pour the remaining alcohol in the sink, dispose of the cup in the garbage. Step 24: Wipe down work surface with 1:9 bleach water solution. Step 25: Identify a location in your home where the inoculated agar plates can incubate agar-side up (inverted) for approximately 48 hours. The location should be room temperature (21°C-25°C), away from heating or air-conditioning vents, out of direct sunlight, and secure from children and pets. An empty cabinet works well. If a countertop is used, place cultures in a box such, as your empty HOL box. Use the

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