Microbiology Task 3

docx

School

Western Governors University *

*We aren’t endorsed by this school

Course

C453

Subject

Biology

Date

Jan 9, 2024

Type

docx

Pages

Uploaded by ChefSandpiperPerson147

Rabeca Plummer Clinical Microbiology Laboratory C453 6/3/2021 Task 3

Introduction In this experiment, I tested the susceptibility of Staphylococcus Epidermidis to three different antibiotics: penicillin, novobiocin, and gentamicin. Testing a patient specific infection is important to ensure that they are receiving the appropriate antibiotics. Broad spectrum antibiotics target many types of bacteria, while narrow spectrum antibiotics target only a few specific ones. (Gerber, 2020) Over time, the human body can become resistant to certain antibiotics, creating MRSA infections. By specifying the narrow spectrum antibiotic used for the infection, healthcare providers can allow for a more targeted attack on the infection and less chance of complication and long-term effect. Unfortunately, culturing bacteria takes days (as show in my experiment) and patients don’t have days to wait before receiving treatment, this is where broad spectrum antibiotics are so important. They are a large-scale attack on the most likely offenders while we await the cultures. Broad spectrum antibiotics allow the patient to start to fight the infection. Therefore, both types of antibiotics have their time and places, but it is important to know which antibiotics the specific infection is susceptible to. Another key feature of antibiotics is selective toxicity, this is the antibiotics’ ability to identify the bacterial infection, target it, and kill it, without being toxic to the human host. (Purssell et al., 2019) This is different than say, chemotherapy, that can damage healthy host cells in the process of killing cancerous ones. My hypothesis for this experiment was that the S. epidermidis would be susceptible to penicillin, susceptible to gentamicin, and susceptible to novobiocin. This will be apparent visually with rings of clear space around each antibiotic disk in the Petri dish. 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 -Antibiotic disc - Gentamicin -Antibiotic disc – Novobiacin -Antibiotic disc – Penicillin -Metric ruler -Plastic tweezers -Sterile swabs 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.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

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: Kirby-Bauer Diffusion Testing Step 1: Gather all supplies from the above list. Step 2: Wash 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. Pour a cup of alcohol for item sterilization. Step 5: Use a permantent marker to divide an agar plate into 3 equal sections. Label the sections “Penicillin”, “Gentamicin” and “Novobiocin”. Step 6: Light the tea candle. Step 7: Gather one sterile swab from its package, do not touch the swab to any surfaces. Step 8: Remove the id from the active S. Epidermidis broth and flame the rim of the tube to sterilize. Step 9: Carefully insert the swab into the culture vial and submerge the tip into the broth. Step 10: Quickly transfer the swab to the agar plate and swab the entire surface of the plate, all the way up to the sides. Step 11: Turn the dish 90 degrees and continue swabbing the entire dish. Step 12: Place the lid on the plate, allow the plate to sit with the lid up until the broth is absorbed by the agar. Step 13: Flame the rim of the culture tube and replace the lid. Step 14: Extinguish the candle. Step 15: Soak the swab in the bleach bucket for 20 minutes before disposing of it in the garbage. Step 16: Place plastic tweezers in the alcohol cup to sterilize them for 20 seconds. Shake to dry. Step 17: Using the sterile tweezers, remove one antibiotic disk from it’s bag and place it onto the agar surface corresponding to the antibiotic name. Step 18: Repeat steps 16 and 17 with the other 2 antibiotics. Step 19: Return the lid to the plate, allow the plate to sit, lid up, for 10 minutes. This will allow the disks to attach to the surface of the agar. Step 20: Place the plate in your incubation location for 48 hours. Step 21: Return the culture vial to your incubation location. Step 22: Wipe the work surface down with 1:9 bleach water solution. Step 23: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water. Step 24: Create a hypothesis based on your knowledge of S. Epidermidis. PART 2 Step 25: Check the agar plate for growth after 48 hours, if no white film has appeared, allow the plate to incubate for an additional 24 hours. Step 26: Wipe the work surface down with 1:9 bleach water solution. Pour a bleach bucket for item disposal.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Step 27: Wash your hands thoroughly with soap and water. Step 28: Put on your PPE; gloves, goggles, apron, and face mask. Step 29: Gather the developed agar plate and a metric ruler. Step 30: Without removing the lid, measure the zone of inhibition around each disk by placing the ruler over the center of each disk and measuring the area around the disk with no growth. Record your results. Step 31: Place the agar plate in the bleach bucket for 20 minutes before disposing of it in the garbage. Step 32: Wipe the work surface down with 1:9 bleach water solution. Step 33: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water. Results After 72 hours of incubation, my agar plates showed no growth around the antibiotic disks. This implied that the strain of S. Epidermidis that I had cultured was very susceptible to all three antibiotics; penicillin, gentamicin, and novobiocin. Discussion The lack of growth on my agar plate in any sections lead me to believe that I was unsuccessful in transferring enough of the S. Epidermidis bacteria to my agar plate in the first part of the experiment. If we are going by the information obtained by my experiment’s results, all three antibiotics would be reasonable to use in this scenario to treat this infection. Resources Purssell, E. (2019, September 4). Antimicrobials . Understanding Pharmacology in Nursing Practice . https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120529/ . Gerber, J. (2020, December 2). Comparing Broad- and Narrow-Spectrum Antibiotics for Children with Ear, Sinus, and Throat Infections. https://www.pcori.org/research-results/2013/comparing-broad-and-narrow-spectrum- antibiotics-children-ear-sinus-and-throat .

Image 1: Escherichia Coli Growth

Image 2: Saccharomyces Cerevisiae Growth

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Image 3: Staphylococcus Epidermidis Growth

Image 4: Antibiotic susceptibility photo 1 Image 5: Antibiotic susceptibility photo 2

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Microbiology Task 3

Related Documents