Antimicrobial Susceptibility Testing

Antimicrobial
Susceptibility
Testing
Rochelle D. Darlucio, RMT, MPH
Pamela Sengson-Sevilla, RMT, ASCPi
College of Medical Laboratory Science
Our Lady of Fatima University
DEFINITIONS:
Ø ANTIMICROBIALS are compounds that kill or

inhibit microorganisms.
Ø ANTIBIOTICS are antimicrobials, usually of low

molecular weight, produced by microorganisms
that inhibit or kill other microorganisms.
Antimicrobial Susceptibility Testing
Ø Performed on bacteria and fungi isolated from clinical specimens to

determine which antimicrobial agents might be effective in treating
infections caused by these organisms
Ø PRIMARY GOAL: to determine whether the bacterial isolate is capable of

expressing resistance to the antimicrobial agents selected for treatment
Ø Often performed by a disk diffusion or dilution (minimal inhibitory

concentration [MIC]) method.
Ø Standards that describe these methods are published and frequently updated
by the Clinical and Laboratory Standards Institute (CLSI), formerly the
National Committee for Clinical Laboratory Standards [NCCLS]).
The standardized components of antimicrobial
susceptibility testing include:
Ø Bacterial inoculum size
Ø Growth medium (typically a Mueller-Hinton base)
Ø pH
Ø Cation concentration
Ø Blood and serum supplements
Ø Thymidine content
Ø Incubation atmosphere
Ø Incubation temperature
Ø Incubation duration
Ø Antimicrobial concentrations
Reasons and Indications for Performing Antimicrobial
Susceptibility Tests
Ø Antimicrobial susceptibility testing should be performed on a

bacterial isolate from a clinical specimen if the isolate is
determined to be a probable cause of the patient’s infection and the
susceptibility of the isolate to particular antimicrobials cannot be
reliably predicted based on previous experience with the bacteria
at a specific health care facility.
Ø Susceptibility testing of isolates can also provide information on

decreases in the susceptibility of bacteria to antimicrobials
Factors to Consider When Determining Whether Testing Is
Warranted
Ø Body site from which the bacterium was isolated

Ø Presence of other organisms and quality of the
specimen from which the organism was grown
Ø Host’s status
Traditional Antimicrobial Susceptibility Test Methods
1. DISC STORAGE
> Long term storage: -20oC or below in a non–frost-free freezer
> A working supply of disks can be stored in a refrigerator at 2°C to 8°C for at least 1
week.
> Disks should always be stored in a tightly sealed container with desiccant.
> The container should be allowed to warm to room temperature before it is opened
to prevent condensation
Ø Inoculum Preparation and Use of McFarland Standards
Ø Inoculum Preparation
Ø one of the most critical steps in susceptibility testing.
Ø Prepared by adding cells from four to five isolated colonies of similar colony morphology
growing on a non-inhibitory agar medium to a broth medium and then allowing them to
grow to the log phase
Ø Can also be prepared directly by suspending colonies grown overnight on an agar plate
directly in broth or saline.
Ø This direct inoculum suspension preparation technique is preferred for bacteria that grow
unpredictably in broth. Because it does not rely on growth in an inoculum broth, the use of
fresh (16- to 24-hour) colonies is imperative.
Ø Use of a standard inoculum size is as important as culture purity and is accomplished by

comparing the turbidity of the organism suspension with a turbidity standard.
Ø Inoculum Preparation and Use of McFarland

Standards
Ø McFarland Turbidity Standards

Ø False-susceptible results may occur if too few
bacteria are tested, and false-resistant results may
be the outcome of testing too many bacteria.
Ø McFarland standards can be prepared by adding

specific volumes of 1% sulfuric acid and 1.175%
barium chloride
Ø McFarland 0.5 standard

Ø 99.5 mL of 1% sulfuric acid
Ø 0.5 mL of 1.175% barium chloride.
Ø Inoculum Preparation and Use of McFarland Standards

Ø Inoculum Standardization
Ø the inoculated broth or direct suspension is vortexed thoroughly.
Ø Under adequate lighting, the tube is positioned side by side with the
McFarland 0.5 standard against a white card containing several
horizontal black lines
METHODS OF Antimicrobial Susceptibility Testing:
A. DIFFUSION METHOD
1. Kirby-Bauer Diffusion Method (most
common)
2. Agar Cup Diffusion Method
3. Agar Cylinder Diffusion Methods
4. Epsilometer or Gradient Diffusion Method
B. DILUTION METHOD
1. Macrobroth Method or Tube Dilution Method
2. Microtube Dilution Method
KIRBY-BAUER METHOD
1. Also known as Agar Diffusion Method or Disk
Diffusion Method
-used to determine the sensitivity or resistance
of a bacterium to an antimicrobial.
1. Principle:
- A standardized suspension of organism is inoculated into
MHA (Mueller-Hinton Agar)
- Paper disk impregnated with specific antibiotics
concentration are placed into the agar
- After 16-20 hours incubation, the diameters of the zone
of inhibitions are measured
- Results are compared to determine susceptibility or
resistance
KIRBY-BAUER METHOD
STEP-BY-STEP PROCEDURE:
1. Preparation of pure inoculum, using any of the following:

a) Mueller-Hinton Broth
b) Trypticase Soy Broth
c) Sterile Distilled Water
d) Natural Saline Solution
e) Brain Heart Infusion Broth
2. Standardize pure inoculum, using 0.5 McFarland Standard

i. If standard more turbid than inoculum à add colonies to
inoculum or incubate inoculum
ii. If inoculum more turbid than standard à add distilled
water to inoculum
KIRBY-BAUER METHOD
3. Streak the pure inoculum into the
medium (MHA)
i. Use a sterile cotton swab and

streak with no space in between
KIRBY-BAUER METHOD
KIRBY-BAUER METHOD
4. Apply antibiotic discs
i. Using forceps aseptically

ii. Space – at least 15mm each antibiotic
5. Incubate
i. Normally 35°C for 16-20 hours.

6. Measure the zone of inhibition
i. Instrument: ruler or microcaliper

ii. Unit: mm
KIRBY-BAUER METHOD
Definitions of Susceptibility Testing
Interpretive Categories
Susceptible (S)
Indicates that the antimicrobial agent in question may be an appropriate choice for
treating the infection caused by the organism. Bacterial resistance is absent or at a clinically
insignificant level.
Intermediate (I)
Indicates a number of possibilities, including:
• The potential utility of the antimicrobial agent in body sites where it may be concentrated (e.g.,
the urinary tract) or if high concentrations of the drug are used
• Possible effectiveness of the antimicrobial agent against the isolate, but possibly less so than
against a susceptible isolate
• Use as an interpretive safety margin to prevent relatively small changes in test results from
leading to major swings in interpretive category (e.g., resistant to susceptible or vice versa)
Resistant (R)
Indicates that the antimicrobial agent in question may not be an appropriate choice for
treatment, either because the organism is not inhibited with serum-achievable levels of the drug
or because the test result highly correlates with a resistance mechanism that indicates
questionable successful treatment.
Dilution Susceptibility Testing Methods
ü used to determine the MIC
ü Various concentrations of an antimicrobial agent

are added to broth or agar media.
ü organism is interpreted as nonsusceptible,

susceptible, intermediate, or resistant to each
agent
ü Antimicrobial Stock Solutions
ü must be prepared from reference standard antimicrobial

powders, not from the pharmaceutical preparations
administered to patients
ü frozen in non–frost-free freezers
ü Optimal temperature: below −60° C , −20° C storage is

acceptable for some agents
ü Broth Macrodilution (Tube Dilution) Tests
ü a twofold serial dilution series, each containing 1 to 2 mL of antimicrobial agent, is

prepared
ü Mueller-Hinton broth
ü A standardized suspension of test bacteria is added to each dilution to obtain a final

bacterial concentration of 5 × 105 CFU/mL.
ü A growth control tube (broth plus inoculum) and an uninoculated control tube (broth
only) are used with each test.
ü After overnight incubation at 35° C, the MIC is determined visually as the lowest
ü Broth Microdilution Tests
ü Plastic trays contain between 80 and 100 (usually 96) wells.
ü Wells are filled with small volumes (usually 0.1 mL) of twofold dilution concentrations of antimicrobial
agent in broth.
ü .An intermediate dilution of this inoculum suspension is prepared in water or saline, and a multipronged
inoculator or other type of inoculating device is used to inoculate the wells to obtain a final concentration of
approximately 5 × 105CFU/mL (5 × 104 CFU/0.1-mL well).
ü A growth control well and uninoculated control well are included on each tray.
ü After overnight incubation at 35° C, the tray is placed on a tray-reading device to

facilitate visual examination of each well
ü Agar Dilution Tests
ü Specific volumes of antimicrobial solutions are dispensed into premeasured volumes of
molten and cooled agar, which is subsequently poured into standard Petri dishes
ü A series of plates containing various concentrations of each antimicrobial agent and

growth control plates without antimicrobial agent are prepared.
ü The agar is allowed to solidify, and then a standard number of test bacteria (104 CFU for
aerobes) are spot inoculated onto each plate using a multipronged replicating device
ü After overnight incubation, the MIC is read as the lowest concentration of antimicrobial
agent that inhibits the visible growth of the test bacterium (one or two colonies are
ignored)

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Antimicrobial

Ø ANTIMICROBIALS are compounds that kill or

Ø ANTIBIOTICS are antimicrobials, usually of low

Ø Performed on bacteria and fungi isolated from clinical specimens to

Ø PRIMARY GOAL: to determine whether the bacterial isolate is capable of

Ø Often performed by a disk diffusion or dilution (minimal inhibitory

Ø Blood and serum supplements

Ø Antimicrobial susceptibility testing should be performed on a

Ø Susceptibility testing of isolates can also provide information on

Ø Body site from which the bacterium was isolated

Ø one of the most critical steps in susceptibility testing.

Ø Use of a standard inoculum size is as important as culture purity and is accomplished by

Ø Inoculum Preparation and Use of McFarland

Ø McFarland Turbidity Standards

Ø McFarland standards can be prepared by adding

Ø McFarland 0.5 standard

Ø Inoculum Preparation and Use of McFarland Standards

Ø the inoculated broth or direct suspension is vortexed thoroughly.

1. Preparation of pure inoculum, using any of the following:

2. Standardize pure inoculum, using 0.5 McFarland Standard

i. Use a sterile cotton swab and

4. Apply antibiotic discs

i. Using forceps aseptically

i. Normally 35°C for 16-20 hours.

i. Instrument: ruler or microcaliper

ü Various concentrations of an antimicrobial agent

ü organism is interpreted as nonsusceptible,

ü must be prepared from reference standard antimicrobial

ü frozen in non–frost-free freezers

ü Optimal temperature: below −60° C , −20° C storage is

ü a twofold serial dilution series, each containing 1 to 2 mL of antimicrobial agent, is

ü A standardized suspension of test bacteria is added to each dilution to obtain a final

ü After overnight incubation at 35° C, the tray is placed on a tray-reading device to

ü A series of plates containing various concentrations of each antimicrobial agent and

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