ACTIVITY 11

ANTIMICROBIAL SENSITIVITY TESTING

Introduction

Modern medicine is dependent on chemotherapeutic agents. These

agents are used to treat certain diseases. They are capable of destroying
pathogenic microorganisms and inhibit their growth at concentrations low
enough to avoid undesirable damage to the host. Determination of
antimicrobial effectiveness against specific pathogen is essential to proper
therapy. Testing can show which agents are the most effective against a
pathogen.

This activity introduces antimicrobial sensitivity testing of bacteria using

the Kirby-Bauer and broth dilution methods.

Learning Objectives
At the end of the activity, the students are expected to:
1. perform the Kirby-Bauer and broth dilution methods in testing the
effectivity of chemotherapeutic agents.
2. interpret the Kirby-Bauer sensitivity test plate using the zone of
inhibition.
3. identify the resistance and susceptibility of the tested bacteria to different
antibiotics.
4. differentiate broad spectrum from narrow spectrum drug.

Materials

Antimicrobial disks
Sterile tubes Pipettor with tips
Forceps Sterile swabs

Reagents and Culture Media

Mueller-Hinton agar plates Mueller-Hinton broth
Sterile saline solution (5 ml each)
McFarland No. 0.5 turbidity standard

Cultures
Staphylococcus aureus Pseudomonas aeruginosa
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 Escherichia coli Bacillus cereus

Procedures

A. Agar Disk Diffusion Method

1. Touch 4 to 5 colonies of S. aureus with sterilized and cooled
inoculating loop. Emulsify the colonies in 5 ml of sterile saline until
the turbidity is approximately equivalent to that of 0.5 McFarland
standard.
2. Dip a swab into the bacterial suspension, express any excess fluid
against the side of the tube, and inoculate the surface of MHA plate
as follows: first streak the whole surface of the plate closely with the
swab; then rotate the plate through a 45° angle and streak the whole
surface again; finally rotate the plate another 90° and streak once
more.
3. Discard the swab in disinfectant.
4. Repeat steps 1 and 2 with K. pneumoniae, E. coli and P. aeruginosa
broth culture on nutrient agar plates.
5. Heat the forceps in alcohol lamp and allow to cool.
6. Pick up an antimicrobial disk with the forceps and place it on the agar
surface of the inoculated plates.
7. Press the disk gently into full contact with the agar, using the tips of
the forceps.
8. Heat the forceps again and cool.
9. Repeat steps 5 and 7 until about six different disks are on one plate,
spaced evenly away from each other.
10. Invert the plates and incubate them at 37°C for 18 to 24 hrs.
11. After 24 hrs, measure the zone of inhibition. Answer items 1-3 in
your worksheet. (Refer to Appendix A)

B. Broth Dilution Method

1. Place nine sterile tubes in a rack and label them accordingly.
2. With a 1 mL pipettor, place 0.5 ml of sterile broth to each tube
(except tube 1).
3. Place 1 mL of of ampicillin to the first tube (Figure 11.1). Discard the
pipette tip. The concentration of ampicillin in this tube is 2 µg/mL.
4. Take a fresh pipette tip, get 0.5 mL inoculum from the first tube and
transfer into the second tube to have 1 µg/mL concentration. Discard
the pipette tip.
5. With a fresh pipette, mix the contents of the second tube and
transfer 0.5 ml to the third tube (0.25 µg/mL).
6. Continue the dilution process through tube number 7. The eighth and
ninth tubes receive no antibiotic.
7. After the contents of the seventh tube are mixed, discard 0.5 ml of
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 broth so that the final volume in all tubes is 0.5 ml.
8. From the plate culture of E. coli prepare a suspension of the
organism in 5 ml of saline equivalent to a 0.5 McFarland standard.
9. With a sterile 1-ml pipettor, transfer 0.1 ml of E. coli suspension into
tubes 1 to 8. Tube no. 8 will serve as growth control, while tube no.
9 will serve as broth control.
10. Shake the rack gently to mix the tube contents and place the
tubes in the incubator for 18 to 24 hours.
11. Repeat steps 1 to 10 using K. pneumoniae, S. aureus and P.
aeruginosa
12. After 24 hrs, observe for the presence of growth. Answer items
4-5 in your worksheet.

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Figure 11.1. Broth dilution method for measuring minimum inhibitory
concentration
Camacho, Kate Frances G.
CAMACHO, Kate Frances G.
Name Score
Group 3 DateMay 18, 2020

WORKSHEET 11
ANTIMICROBIAL SENSITIVITY TESTING

1. Fill in Table 11.1. (5 pts)

Table 11.1. Zone of inhibition of the test antibiotics against selected

bacteria.
Bacteria Antibiotics
P A Pb S T Q
S. aureus 29 29 7 7 24 9
E. coli 10 9 14 21 23 29
P. aeruginosa 6 26 16 15 19 15
S. typhi 11 6 17 26 19 32

2. Interpret the results based on the resistance or sensitivity of bacteria

against antibiotics (Refer to Performance Standards for Antimicrobial Disk
Susceptibility Tests—11th Informational Supplement (M100-S11).
National Committee for Clinical Laboratory Standards, NCCLS, 2001). (4
pts)

The antibiotics Penicillin, Ampicillin, and Tetracycline are susceptible. While the rest of the antibiotics are
S. aureus resistant which means bacteria can grow if these drugs are present.
The antibiotics Penicillin and Ampicillin are resistant to the bacterium. While the rest of the antibiotics are
susceptible which means bacteria can’t grow if drug is present.
E. coli
The antibiotics Penicillin is the only resistant which means bacteria can grow if drugs are present. While it is
susceptible to the rest of the antibiotics given.
P. aeruginosa _
The antibiotics Penicillin and Ampicillin are resistant, and the rest of the antibiotics are susceptible to the
bacteria.
S. Typhi

3. Of the antibiotics used, which can be considered as broad spectrum

antibiotic/s? Provide an explanation. (3 pts)
_
Based on the results, tetracycline can be considered as broad spectrum antibiotics because all the

given bacteria are sensitive to this antibiotic. It 65

exerts a bacteriostatic effect on bacteria by binding

reversible to the bacterial 30S ribosomal subunit, and blocking incoming aminoacyl tRNA from

binding to the ribosome acceptor site.

 _

4. Fill in the table below with (+) if there is the presence of growth and (–) if
otherwise. (5 pts)

Table 11.2. Minimum inhibitory concentration of ampicillin against the

test bacteria.
Test Bacteria Titer
2 4 8 16 32 64 128
K. pnemoniae + + + + + + +
S. aureus + + + + + + +
P. aeruginosa - - - - - - -
E. coli - - + + + + +

5. What is the MIC of the antibiotic used? (4 pts)

S. aureus _ None – ug/ml = resistant to ampicillin

0.5 ug/ml
E. coli
<0.015 ug/ml (growth was not seen up until 128 titer)
P. aeruginosa _
Acc. to my research it is resistant like S. aureus
K. pneumoniae

Conclusion
_Sensitivity testing is used to determine the right antibiotic treatment for an infection and to monitor
Changes in bacterial resistance to antibiotics. In this activity, the students were able to characterize the

Drug effectiveness if they are susceptible, intermediate and resistant. Also, the students were able to

Differentiate the broad spectrum from narrow spectrum drug.

References

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_Sensitivity Analysis: Purpose, Procedure, and Results. (2020). Retrieved 21 May 2020, from
https://www.healthline.com/health/sensitivity-analysis

What are sensitive, a. (2020). What are sensitive, intermediate, and resistant antibiotics?

| Socratic. Retrieved 21 May 2020, from https://socratic.org/questions/what-are-sensitive-intermediate-

and-resistant-antibiotics

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