Yinyang Ackerman
BACTE LAB
Module 9: Antimicrobial Susceptibility Testing
● Measure the ability of an antimicrobial agent to
inhibit bacterial growth in vitro.
● GOALS:
○ Detect possible drug resistance in
common pathogens
○ Assure susceptibility to drugs of choice
for particular infection
● TWO GENERAL METHODS:
○ According to action:
■ Diffusion
■ Dilution
○ According to the type of media used:
■ Broth
■ Agar
DILUTION METHOD
- Various amounts of antimicrobial
substances incorporated into liquid or
solid media followed by inoculation of
test bacteria.
DIFFUSION METHOD
- Place a filter disc/porous cup/bottomless
cylinder containing measured quantity of drugs
on the solid media that has been inoculated
with test bacteria.
Methods:
1. Disk Diffusion Method (Kirby-Bauer Test)
2. Broth Dilution Test
3. Agar Dilution Test
4. Epsilometer Test (E-Test)
1. Disk Diffusion Method (Kirby-Bauer Test)
- a.k.a . KIRBY-BAUER METHOD
- Standardized inoculum of the organism
is swabbed on an MHA (Mueller-Hinton
Agar) plate;
- Antimicrobial discs are placed on the
surface
- Standard inoculum size 1.5 x 10^8
CFU/mL (prepare suspension)
- Zones of inhibition are then measured
after 24 hr incubation at 37 degrees
celsius.
- Zones are then interpreted as
susceptible, intermediate or resistant.
PROCEDURE:
1. Select colonies
2. Prepare inoculum suspension
3. Standardize inoculum
suspension
4. Inoculate plate
5. Add antimicrobial disks
6. Incubate plate
(incubation:18-24 hrs)
7. Measure zones of
Inhibition (clear zone without
bacterial growth)
8. Interpret results
PREPARATION OF INOCULUM SUSPENSION
- Prepare sterile NSS solution which is going to be
inoculated with the colonies picked from the
isolation media.
- The turbidity of the suspension must be
standardized matching the 0.5 McFarland
standard.
- Suspension should be used within 15 minutes.
PRIOR TO PLATE INOCULATION
- Antibiotic disks & MHA plates should be at room
temp. For 1-2 hours.
- (antimicrobials are stored in the refrigerator to
preserve integrity: 2-8 degrees celsius; average:
4 degrees celsius)
- Mix the inoculum suspension thoroughly
- Use a fresh, sterile cotton-tipped swab into the
suspension.
- Remove the excess liquid from the swab by
pressing it against the side of the tube.
INOCULATING THE PLATE
- Inoculate the top of the MHA plate using a swab
by streaking back and forth from edge to edge.
- Rotate the plate approximately 60 degrees
celsius.
APPLYING THE ANTIMICROBIAL DISKS
- Apply the disks within 15 minutes of inoculating
the MHA plate.
- Press each disk down firmly to ensure complete,
level contact with the agar.
- Incubate at 37 degrees celsius for 18-24 hrs.
- Distance from each disk: ≥ 30 𝑚𝑚
- Distance from the edge of the plate: ≥ 10 𝑚𝑚
Points to Remember!!!
Do not use disks beyond their expiration date
Do not store disks in a frost-free freezer
Use FDA cleared products
Use disks with the content specified in NCCLS
standards
Do not relocate a disk once it has touched the
agar surface
INCUBATING THE PLATE
- Incubate plates with agar side up (inverted-to
avoid moisture to build up )
Yinyang Ackerman
PROTEUS SWARMING
MEASURING ZONES: - Measure the obvious zone. Ignore the swarm
even if it covers the zones.
- Trimethoprim-Sulfamethoxazole

SOURCES OF ERROR
REFLECTED LIGHT ● Smaller zones of inhibition
○ Medium is too deep
○ Too dense inoculum
○ Presence of moisture
● Larger zones of inhibition
○ Medium is too shallow
○ Too light inoculum
○ Too dry agar surface
● INTERPRETING:
○ Disks placed too close together
■ Overlapping zones
○ Disks not pressed on agar surface
■ Irregular shaped zones
UNUSUAL ZONES ○ Colonies within zones
- Some zones may be difficult to measure ■ Mixed culture/resistant strains
- Double zone: measure the innermost zone ○ Zone within zone
■ Proteus spp. Swarming

INTERPRETATION OF ZONES
- Diameters of zones are measured in mm using a
ruler or caliper.
- Measurements are then interpreted as either
susceptible, intermediate or resistant basing on
the CLSI standards.
- NOTE: different antibiotics are used for
sensitivity depending on the microorganism that
has grown from the media.
-
Yinyang Ackerman

2. Broth Dilution Method Advantage/Disadvantage

- Various conc. of an Antimicrobial drug
are inoculated with a standard
suspension of test bacteria.
- MIC (Minimum Inhibitory Conc.) is
determined by observing the lowest
conc. of the drug that will inhibit visible
growth of the test bacteria.
- Use Mueller-Hinton Broth
(MHB)
- Preferred method: serial
two-fold dilution conc.
(ug/mL) (e.g. by two)
- This method can be done by
using the test tube dilutions
(macrodilution) or the
plastic microtiter plates
(microdilution)

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MIC VS MBC INTERPRETATION OF RESULTS
● MIC: Minimum Inhibitory Conc. - Measurements are then interpreted as either
○ The lowest conc. of the drug that susceptible, intermediate or resistant basing on
prevents the growth of bacteria the CLSI standards.
● MBC: Minimum Bactericidal Conc. - NOTE: different antibiotics are used for
○ The lowest conc. of the drug that kills sensitivity depending on the microorganism that
99.9% of the organism has grown from the media.

3. Agar Dilution Method

- The organism and the microbial
solutions are brought together on a
culture medium
- Used only in research studies because
plate preparation is laborious
- Uses Mueller-Hinton Agar
- Standard Inoculum size: 5𝑥104 CFU/mL
- Agar dilutions are most often prepared
in petri dishes. It would be convenient
to use 90 mm diameter
- petri dishes and add one ml of desired
drug dilutions to 19 ml of broth.
- The factor of agar dilution must be
allowed for in the first calculation as
follows.
Yinyang Ackerman

PROCEDURE:
1. Standardize an inoculum - Can be classified as antibacterial, antifungal,
2. W/in 15 minutes after standardization, dip a antiprotozoal, or as antiviral …
sterile swab into the TSB.
3. Streak the entire agar surface of a plate (MHA)
three times (3x), turning the plate 60˚ between
streaking to obtain even inoculation.
4. Allow plate to dry completely at room
temperature for 3 minutes but not longer than
15 minutes.
5. Take the cork borer (diameter of 8 or 10mm) all
this time immersed in an Erlenmeyer flask
containing ethanol. Shake off the alcohol and
sterilize over an alcohol flame. Cool in air for 20
seconds. IDEAL ANTIMICROBIAL AGENT:
6. Stab the agar all the way down to the bottom of 1. Exhibits selective toxicity.
petri plates with the cork borer held upright and 2. Kills or inhibits the growth of pathogens.
slowly pull up the borer to create a well cleanly 3. Causes no allergic reaction to the host.
cut hole from the plate. 4. Stable when stored in either solid or liquid
7. Deliver the corresponding stock dilution into the forms.
respective agar wells using a micropipettes or 5. Remains in specific tissues in the body long
syringe until the entire hole is filled up but not enough to be effective.
overflowing. 6. Kills the pathogens before they mutate and
8. Use one of the wells to deliver the positive become resistant to it.
control.
9. Incubate plates in the usual position not MECHANISM OF ACTION
inverted for 18 to 24 hours 37°C.
10. Observe and measure zone of inhibition.

4. Epsilometer Test
- a rectangular plastic strip device that
contains predefined gradient of
antibiotic concentrations that
corresponds to MIC dilutions
- Elliptical zone of inhibition of bacterial
growth is seen around the test strip.
The zone edge intersects the plastic
strip at a specific level corresponding to
the inhibitory concentration of the drug
that inhibits the microorganism.

ANTIMICROBIAL AGENTS
- Any chemical or drug used to treat infectious
disease by inhibiting or killing pathogens in vivo.
- Synthetic drugs are manufactured in the
laboratory either extracted derived from
chemicals or antibiotics.
- Antibiotic is a substance produced by a microbe
that is effective in killing other microbes.