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Name Levi Ianu P. Pepaña Section A1A56 Date Submitted 10/18/2021

LABORATORY EXERCISE 6
ANTIBIOTIC SUSCEPTIBILITY TESTING:
The Virtual Kirby-Bauer Method

WORKSHEET

Refer to the Module Instructions in Kirby Bauer as guide.

Table 1. Escherichia coli Zone of Inhibition (ZOI) Measurement

Zone of Interpretation
Screenshot of ZOI measurement Inhibition (Resistant, Intermediate,
diameter (in mm) or Susceptible)
a. Amoxicillin (AMC)

11 mm Resistant

b. Cephalothin (CF)

18 mm Susceptible

c. Chloramphenicol (C)

30 mm Susceptible
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d. Ciprofloxacin (CIP)

32 mm Susceptible

e. Clindamycin (CC)

6 mm Resistant

f. Erythomycin (E)

7 mm Resistant

g. Oxacillin

6 mm Resistant

h. Penicillin G (P)

6 mm Resistant
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i. Streptomycin (S)

20 mm Susceptible

j. Tetracycline (TE)

18 mm Intermediate

k. Tobramycin (TM)

24 mm Susceptible

l. Trimethopim Sulfa (SXT)

21 mm Susceptible
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Table 2. Pseudomonas aeruginosa Zone of Inhibition (ZOI) Measurement

Zone of Interpretation
Screenshot of ZOI measurement Inhibition (Resistant, Intermediate,
diameter (in mm) or Susceptible)
a. Amoxicillin (AMC)

7 mm Resistant

b. Cephalothin (CF)

6 mm Resistant

c. Chloramphenicol (C)

15 mm Intermediate

d. Ciprofloxacin (CIP)

37 mm Susceptible
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e. Clindamycin (CC)

6 mm Resistant

f. Erythomycin (E)

6 mm Resistant

g. Oxacillin

6 mm Resistant

h. Penicillin G (P)

6 mm Resistant

i. Streptomycin (S)

5 mm Resistant
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j. Tetracycline (TE)

6 mm Resistant

k. Tobramycin (TM)

19 mm Susceptible

l. Trimethopim Sulfa (SXT)

6 mm Resistant
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Table 3. Staphylococcus aureus Zone of Inhibition (ZOI) Measurement

Zone of Interpretation
Screenshot of ZOI measurement Inhibition (Resistant, Intermediate,
diameter (in mm) or Susceptible)
a. Amoxicillin (AMC)

7 mm Resistant

b. Cephalothin (CF)

6 mm Resistant

c. Chloramphenicol (C)

6 mm Resistant

d. Ciprofloxacin (CIP)

5 mm Resistant
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e. Clindamycin (CC)

6 mm Resistant

f. Erythomycin (E)

7 mm Resistant

g. Oxacillin

6 mm Resistant

h. Penicillin G (P)

7 mm Resistant

i. Streptomycin (S)

6 mm Resistant
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j. Tetracycline (TE)

6 mm Resistant

k. Tobramycin (TM)

5 mm Resistant

l. Trimethoprim Sulfa (SXT)

5 mm Resistant

m. Vancomycin (VA)

16 mm Susceptible
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GUIDE QUESTIONS:
Note: Your answers should be brief and concise. Your answers must not exceed the lines provided.

1. Which chemotherapeutic agent was the most effective and least effective to each test
organism (E. coli, P. aeruginosa, and S. aureus), respectively?
E. coli – Ciprofloxacin (most effective), Clindamycin, Oxacillin, Penicillin (least effective);
P. aeruginosa – Ciprofloxacin (most), Streptomycin (least); S. aureus – Vancomycin (most),
Ciprofloxacin, Tobramycin, Trimethoprim Sulfa (least).

2. Is the agar diffusion technique measuring bacteriostatic or bactericidal activity?

The agar diffusion technique is measuring bacteriostatic because it measures the diameter of
the zone of inhibited growth around the disk.

3. In laboratory settings, what factors affect the zone of inhibition?

Multiple factors that affect the zone of inhibition includes the drug concentration on the disk,
drug solubility, rate of drug diffusion through the agar, and the thickness of the agar.

4. In which growth phase is the organism most sensitive to the antibiotic?

The log (exponential) phase is where microorganisms are most sensitive or susceptible to
antibiotics.

5. What are other methods of measuring the sensitivity of organisms to antibiotic?

Other methods are agar dilution, broth macro and microdilution, and concentration gradient
test.

6. What is MIC and MBC? How are they measured?

MIC (Minimum Inhibitory Concentration) inhibits the growth of microorganism measured

after overnight incubation while MBC (Minimum Bactericidal Concentration) kills the
microorganism and is measured by subculturing broths used for MIC to fresh agar plates.

7. What is being measured in the agar-disk diffusion method, bactericidal or bacteriostatic

activity of the organism?

The agar-disk diffusion method is measuring the bacteriostatic activity of the microorganism
because it measures the diameter of the zone of inhibited growth around the disk.

8. Why is the agar-disk diffusion method not a perfect indication on how well the
chemotherapeutic agent will perform in vivo?
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The agar-disk diffusion method doesn’t give perfect indication because some microorganisms
such as fastidious ones, mycobacteria and fungi have special growth requirements or special
technique for susceptibility testing. If not, results may be unexpected or borderline.

9. What is a McFarland Standard? How is it useful to antibiotic sensitivity test?

A McFarland Standard is a chemical solution from the reaction of barium chloride and
sulfuric acid. It is used in AST to standardize the approximate number of bacteria in a liquid
suspension by comparing the tests’ turbidity.

10. Why is Mueller Hinton Agar used in this test and not other ordinary agar media?

Mueller Hinton Agar is specifically used because it is non-selective and non-differential

(most can grow), contains starch that absorbs toxins so it cannot interfere with the antibiotic,
and is a loose agar meaning it allows better diffusion leading to truer zones of inhibition.