Gannon Antibiotic Susceptibility Testing of Staphylococcus Lab Report Complete the Bacterial Identification Virtual Lab using the following URL https://www
Gannon Antibiotic Susceptibility Testing of Staphylococcus Lab Report Complete the Bacterial Identification Virtual Lab using the following URL https://www.hhmi.org/biointeractive/bacterial-identification-virtual-lab and the followingBacterial ID WS Microbiology
Lab 10
ANTIBIOTIC SUSCEPTIBILITY TESTING OF AN ISOLATE OR STAPHYLOCOCCUS
Read Atlas Section 19
Antibiotic resistance is a growing health issue. In this exercise you will be isolating your own
staphylococci from skin, nose, or ear, using a selective growth medium. The staphylococcal
isolates will then be evaluated against a number of antibiotics.
Alternatively, you will be given a culture that can be utilized for the determining its antibiotic
susceptibility. Reference: Omoto, C. K. and K. Malm. 2003. The American Biology Teacher
65: 133-135
Materials
Sterile saline
Sterile cotton swabs
Mannitol salt agar plates
Trypticase soy agar plates or
Nutrient agar plates or
Antibiotic discs
Forceps with 95% alcohol or sterilized forceps
Control cultures of Staphylococcus
Ruler
Procedure
Day 1: Using a sterile cotton swab and sterile saline as a wetting agent, swab an area of your
skin and streak the mannitol salt agar plate. Incubate the plate at 37 C until the next class
period.
Day 2: Observe the plates for colonies. Record any non-mannitol fermenting colonies versus
mannitol fermenting colonies. Pick desired colonies and transfer to an agar slant, at the same
time transfer some colonies to a nutrient broth tube. Incubate slants and tubes at 37C
overnight.
Day 3: Streak culture from the nutrient broth tube onto a trypticase soy agar plate. Make sure
that you cover the whole surface of the plate by going back and forth over the entire surface
area to produce a lawn. Allow the surface moisture to be absorbed by leaving the cover of the
plate ajar for 3 to 5 minutes. Using sterile technique and forceps place antibiotic discs at least
one inch apart and no less than ½ inch from the edge of the agar plate. The disc must be in
firm contact with the agar. To sterilize the forceps, soak them in a beaker of alcohol and then
flame or air dry. Incubate the plates for 24 to 48 hours at 37 C. They can be stored at 4 C for
days before the zone of inhibition is measured.
Day 4: Measure the diameter of the zones of inhibition in mm. Use the interpretive chart for
rating bacteria as resistant, intermediate or susceptible to each of the antibiotics, and determine
the level of resistance of the students bacteria to each antibiotic.
Zone Diameter Interpretive Chart
Zone Diameter
Interpretive Standards (mm)
Antimicrobial Agent
Code
Disc Potency
Resistant
Intermediate
Susceptibleb
Chloramphenicol
Staphylococci
C-30
30 ?g
? 12
13 – 17
? 18
Erythromycin
Staphylococci
E-15
15 ?g
? 13
14 – 22
? 23
Kanamycin
Enterobacteriaceae
and staphylococci
K-30
30 ?g
?13
14 – 17
? 18
Neomycin
N-30
30 ?g
? 12
13 – 16
? 17
NB-30
30 ?g
? 17
18 – 21
? 22
Penicillin h
P-10
10 U
? 28
–
? 29
Streptomycin
Enterobacteriaceae
S-300
S-10
300 ?g
10 ?g
? 11
12 – 14
? 15
Tetracycline ee
Staphylococci
Te-30
30 ?g
? 14
15 – 18
? 19
Novobiocin
f
Watch the following video on setting up disk diffusion assay which is pretty close to how
we would do this in the lab and answer the following questions on the report pages.
Watch the following TED Talk on Factory Farms Antibiotics and Superbugs by Lance
Price and answer the questions on the report pages
Lab Report 10
Page 1
Name
Lab Section
A
B
C
The following data for the diameter of zones of inhibition is provided for each of the antibiotics
tested against Staphylococcus aureus.
Determine from the Zone Diameter Interpretive Chart page 2 above, whether the organism
is sensitive (S), resistant (R) or intermediate (I) in sensitivity.
Test Organism(s) Used
Antibiotics /
Antimicrobials Used
and Concentrations/Units
1.
2.
3.
4.
5.
6.
Penicillin P-10
Neomycin N-30
Streptomycin S-300
Erythromycin E-15
Chloramphenicol C-30
Tetracycline Te-30
Staphylococcus aureus
Zone
(mm)
25
15
17
21
22
20
Sensitive
Classification
Intermediate
Resistant
1. What is the name given to disk diffusion protocol for testing antibiotic resistance?
2. What are the bacteria referred to as they are growing on the plate and why? (Choose
one colony or lawn)
3. What is the clear area around each disk called?
Lab Report 10
Page 2
4. How might a particular bacterium become resistant to an antibiotic like penicillin? Give
two ways
5. How does low dose use of antibiotics contribute to bacterial resistance?
6. How can you help educate the public and bring about changes in meat and antibiotic
consumption?
Microbiology
Lab 10
ANTIBIOTIC SUSCEPTIBILITY TESTING OF AN ISOLATE OR STAPHYLOCOCCUS
Read Atlas Section 19
Antibiotic resistance is a growing health issue. In this exercise you will be isolating your own
staphylococci from skin, nose, or ear, using a selective growth medium. The staphylococcal
isolates will then be evaluated against a number of antibiotics.
Alternatively, you will be given a culture that can be utilized for the determining its antibiotic
susceptibility. Reference: Omoto, C. K. and K. Malm. 2003. The American Biology Teacher
65: 133-135
Materials
Sterile saline
Sterile cotton swabs
Mannitol salt agar plates
Trypticase soy agar plates or
Nutrient agar plates or
Antibiotic discs
Forceps with 95% alcohol or sterilized forceps
Control cultures of Staphylococcus
Ruler
Procedure
Day 1: Using a sterile cotton swab and sterile saline as a wetting agent, swab an area of your
skin and streak the mannitol salt agar plate. Incubate the plate at 37 C until the next class
period.
Day 2: Observe the plates for colonies. Record any non-mannitol fermenting colonies versus
mannitol fermenting colonies. Pick desired colonies and transfer to an agar slant, at the same
time transfer some colonies to a nutrient broth tube. Incubate slants and tubes at 37C
overnight.
Day 3: Streak culture from the nutrient broth tube onto a trypticase soy agar plate. Make sure
that you cover the whole surface of the plate by going back and forth over the entire surface
area to produce a lawn. Allow the surface moisture to be absorbed by leaving the cover of the
plate ajar for 3 to 5 minutes. Using sterile technique and forceps place antibiotic discs at least
one inch apart and no less than ½ inch from the edge of the agar plate. The disc must be in
firm contact with the agar. To sterilize the forceps, soak them in a beaker of alcohol and then
flame or air dry. Incubate the plates for 24 to 48 hours at 37 C. They can be stored at 4 C for
days before the zone of inhibition is measured.
Day 4: Measure the diameter of the zones of inhibition in mm. Use the interpretive chart for
rating bacteria as resistant, intermediate or susceptible to each of the antibiotics, and determine
the level of resistance of the students bacteria to each antibiotic.
Zone Diameter Interpretive Chart
Zone Diameter
Interpretive Standards (mm)
Antimicrobial Agent
Code
Disc Potency
Resistant
Intermediate
Susceptibleb
Chloramphenicol
Staphylococci
C-30
30 ?g
? 12
13 – 17
? 18
Erythromycin
Staphylococci
E-15
15 ?g
? 13
14 – 22
? 23
Kanamycin
Enterobacteriaceae
and staphylococci
K-30
30 ?g
?13
14 – 17
? 18
Neomycin
N-30
30 ?g
? 12
13 – 16
? 17
NB-30
30 ?g
? 17
18 – 21
? 22
Penicillin h
P-10
10 U
? 28
–
? 29
Streptomycin
Enterobacteriaceae
S-300
S-10
300 ?g
10 ?g
? 11
12 – 14
? 15
Tetracycline ee
Staphylococci
Te-30
30 ?g
? 14
15 – 18
? 19
Novobiocin
f
Watch the following video on setting up disk diffusion assay which is pretty close to how
we would do this in the lab and answer the following questions on the report pages.
Watch the following TED Talk on Factory Farms Antibiotics and Superbugs by Lance
Price and answer the questions on the report pages
Lab Report 10
Page 1
Name
Lab Section
A
B
C
The following data for the diameter of zones of inhibition is provided for each of the antibiotics
tested against Staphylococcus aureus.
Determine from the Zone Diameter Interpretive Chart page 2 above, whether the organism
is sensitive (S), resistant (R) or intermediate (I) in sensitivity.
Test Organism(s) Used
Antibiotics /
Antimicrobials Used
and Concentrations/Units
1.
2.
3.
4.
5.
6.
Penicillin P-10
Neomycin N-30
Streptomycin S-300
Erythromycin E-15
Chloramphenicol C-30
Tetracycline Te-30
Staphylococcus aureus
Zone
(mm)
25
15
17
21
22
20
Sensitive
Classification
Intermediate
Resistant
1. What is the name given to disk diffusion protocol for testing antibiotic resistance?
2. What are the bacteria referred to as they are growing on the plate and why? (Choose
one colony or lawn)
3. What is the clear area around each disk called?
Lab Report 10
Page 2
4. How might a particular bacterium become resistant to an antibiotic like penicillin? Give
two ways
5. How does low dose use of antibiotics contribute to bacterial resistance?
6. How can you help educate the public and bring about changes in meat and antibiotic
consumption?
BACTERIAL IDENTIFICATION LAB HANDOUT
PLEASE USE THIS VERSION OF THE WORKSHEET
INTRODUCTION
Go to http://www.hhmi.org/biointeractive/explore-virtual-labs
Scroll down and click on The Bacterial Identification Virtual Lab.
Maximize the screen if you wish. Answer the following questions in the spaces provided.
1. What is the overall purpose of this virtual lab?
2. What are the four basic steps involved in this bacterial identification lab?
3. What is “16S rDNA,” and how is it used to identify species of bacteria?
Click to Enter the Lab. (Click the window on the left-hand side of the screen to enter the lab.) As
you enter the lab, follow the instructions in the lab (left-hand window). Using the information in
the Notebook window on the right, answer the following questions.
PART 1: SAMPLE PREPARATION
4. As the pathology lab technician, what is your task in this virtual lab?
5. Extracting DNA involves which initial step?
6. What is the wire ring used for?
7. Why are the proteolytic enzymes necessary?
8. Why do you then need to inactivate the proteolytic enzymes and how do you do it?
9. After removing the enzymes, why do you spin the sample in the centrifuge?
10. a. What is the pellet?
b. What is the supernatant?
c. Where is the DNA?
PART 2: PCR AMPLIFICATION
Go on to Part 2 and work through the PCR steps. Be sure to read the information in the
notebook, including What is PCR?
11. What does PCR stand for and what is the purpose of PCR?
12. Summarize the process of PCR including the three steps involved in the amplification
Add the Master Mix and answer the following questions:
13. What does the Master Mix contain?
14. What are primers? Why is a primer added?
15. Once the primers bind, what occurs next?
16. What does “highly conserved” mean?
17. Why are highly conserved regions important in this lab?
18. What does “highly variable” mean?
19. Why are highly variable regions important in this lab?
20. What is missing in the negative control tube?
21. What is present in the positive control tube that is not in the negative control tube?
Now run the PCR
Be sure to watch the virtual lab animation before proceeding to the following questions.
22. List each step of a PCR cycle, the temperature, and the duration (time).
a.
b.
c.
23. Describe what happens during each of the steps in one or two sentences.
a.
b.
c.
24. After eight cycles, how many copies of the desired DNA have been synthesized?
25. After 30 cycles?
PART 3: PCR PURIFICATION
26. Approximately how long is the 16s rDNA (bp)?
27. Why would it be useful to run an electrophoresis gel at this point?
28. If you were to run a gel, it would have three lanes. What would each lane contain, and what
would you see in each lane after running the gel?
a.
b.
c.
29. The gel is not run in this virtual lab. In order to purify the PCR product, you use a micro
concentrator column. (Proceed through the virtual lab steps.) What should the final collection
tube contain?
PART 4: SEQUENCING PREPARATION
Click on “Learn about cycle sequencing before proceeding.”
30. Read the first two paragraphs and list the steps in cycle sequencing in the space provided.
Click to go back to Part 4.
31. What do the green and blue tubes contain? Describe the sequencing brew to which
you added your purified PCR.
32. The purpose of the second PCR is not to create identical copies like the first PCR you ran.
What is the purpose of this PCR?
33. Where do scientists obtain primers to be used in PCR and in this technique?
Be sure to watch the virtual lab animation before proceeding to Part 5.
PART 5: DNA SEQUENCING
34. What is the final PCR product, the stuff contained in your 12 tubes?
35. What is the purpose of gel electrophoresis?
36. How do DNA molecules move in relation to charge? Why?
37. What is the purpose of the laser beam in determining a DNA sequence?
Be sure to watch the virtual lab animation before proceeding to Part 6.
PART 6: DNA SEQUENCE ANALYSIS
Click on “Learn about the science behind sequence matching.”
38. What is the ultimate goal of the sequence matching analysis?
39. What is “homology”?
40. What is BLAST and how is it used?
41. Whats a major assumption when drawing evolutionary relationships between organisms
based on DNA sequences?
Click to go back to Part 6 and click on “Learn more about BLAST search results.”
42. Explain what the “Score (bits)” means on an actual BLAST search result.
43. What does an E-value of 3 or less represent?
Click to go back to Part 6 and proceed through the instructions in the right-hand notebook
window.
Hints: “Ctrl A” will select all the data in the pop-up window, “Ctrl C” will copy it, and “Ctrl V”
will paste it into the NCBI website (large yellow box at the top of the BLAST search page).
Follow the steps listed on the page and be patient. BLAST data can take a while to search.
When the BLAST results appear, scroll down below the color key to the significant
alignments, and then go back to the virtual lab window (left) and follow the instructions.
44. What is the scientific name of the bacterium you sequenced?
45. Write a brief description of this bacterium in the space provided.
After completing Sample A, perform DNA sequence analysis on three of the other five samples.
46. Write in the letter of the samples you choose, the scientific name of the bacterium (after
doing a BLAST search), and a brief description of each.
Sample Letter
Bacteria Scientific Name
Brief Description
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