Enclonar, Kimberly / MLS 3A
o 99.5mL of 1% sulfuric acid and 0.5mL of 1.175%
Antimicrobial Susceptibility Testing
November 4, 2020
Marx P. Catalan, RMT, MSMT
Reasons and Indications for Performing AST
• The susceptibility of the isolate to a particular
antimicrobials cannot be reliably predicted
• Provides information on decreases in susceptibility of
bacteria to microbials
Factors to consider when determining whether AST is
warranted
• Body site
o Do not perform AST to organisms that are
anatomically isolated from their normal habitat
o E.g. E. coli in stool, but if E. coli is isolated in
blood, perform AST.
• Presence of other bacteria and quality of specimen
o Do not perform AST in mixed cultures
o It signifies contamination
• Host status
o Perform AST to immunocompromised patients
and patients with antimicrobial allergies
Standardization
Purposes:
• Optimizes bacterial growth conditions
o To remove other environmental limitations
(nutrition and temperature)
• Optimizes conditions for maintaining antimicrobial
integrity and activity
o Ensures that growth inhibition failure is
attributed to organism-associated resistance
mechanism
• Maintains reproducibility and consistency
Standardized Components of AST
1. Bacterial inoculum size
2. Growth medium (Mueller-Hinton Base)
o pH
o Cation concentration
o Blood and serum supplements
o Thymidine content
3. Incubation atmosphere
4. Incubation temperature
5. Incubation duration
6. Antimicrobial concentration
Traditional Antimicrobial Susceptibility Test
Methods
Inoculum Preparation and use of McFarland Standards
Inoculum Preparation
• One of the most critical steps in susceptibility testing
• Prepared by adding cells from 4-5 isolated colonies of
similar colony morphology
• Note: Direct inoculum suspension preparation
technique is preferred for bacteria that are fastidious.
• Tube: NSS
McFarland Turbidity Standards
• Inoculum concentration of bacteria to be tested must
be standardized.
• False susceptible results: too thin inoculum
• False resistance results: too thick inoculum
• 0.5 McFarland Turbidity Standards
o Commonly used standards
barium chloride
o 99.5mL 0.037N H2SO4 and 0.5mL 0.048M BaCl2
o Provides turbidity comparable to bacterial
suspension containing approximately 1.5x10^8
CFU/mL
Inoculum Standardization
• To standardized the inoculum, the suspension should
be examined under adequate lighting.
• The tube is positioned side by side with the 0.5
McFarland Standard against the Wickerham card
(white card with black horizontal lines)
• Note: Once standardized, the inoculum suspensions
should be used within 15 minutes of preparation
• You may also measure the inoculum suspension with
the use of densitometer.
Dilution Susceptibility Testing Methods
• Used to determine the MIC or the lowest
concentration of antimicrobial agent required to
inhibit the growth of the bacterium.
• ↓ MIC, ↑ effective
• Inoculum = 5x10^5
• Broth dilution method for measuring minimum
inhibitory concentration of antibiotics
• One MIC is determined, organism is interpreted as
the following, with the use of CLSI standards
o Nonsusceptible
o Susceptible
o Intermediate
o Resistant
• Broth Macrodilution (Tube Dilution) Tests
o Two-fold serial dilution series containing 1-2mL
of antimicrobial agents
o Standardized suspensions: 5x10^5 CFU/mL
• Broth Microdilution Test
o Miniaturized and adopted to multiwell
microdilution trays
o Plastic trays contain 80-100 (usually 96) wells -
volume: 0.1mL
o Standardized suspensions: 5x10^5 CFU/mL
o Note:
▪ Drug solution in sterile distilled water,
ethanol or DMSO
▪ Drug solution: Dilute 1/10 in MHB
▪ Incubation at 35C for 18hrs
▪ Bacterial suspension: dilute 1/150 in MHB
• Agar Dilution Test
o Shelf-life of agar dilution plates: 1 week stored
at 2-8C
o Reference method for AST of anaerobes and
Neisseria gonorrheae
o Inoculum: 10^4 CFU/mL
Disk Diffusion Testing (Kirby-Bauer Test)
• Most common since 1966
• Uses McFarland Standard, MHB, and paper disk
• Procedure:
o Within 15 minutes of adjusting the turbidity, dip
a sterile cotton swab into the sample streak a
lawn of bacteria on MHA (150mm, 12 disks)
▪ Streak all (no space); rotate 60degrees
o Leave the lid agar 3-5mins (not >15mins to allow
plate to dry)
o Apply antibiotic impregnated disks on the
bacterial lawn
▪ Do not move disk
▪ Using sterile forceps
 Enclonar, Kimberly / MLS 3A
o Incubate for 16-18hrs at 33+- 2C unless Automated Instrument systems
otherwise instructed • BD Phoenix System
o After incubation, observe for a clearing on the • Microscan Walkaway SI
bacterial lawn (zone of inhibition) • TREK Sensititre
o Measure the zone of inhibition using a • VITEK-1, VITEK-2 and VITEK-2 Compact
micrometer caliper or ruler. • Small plastic reagent card
o Use reflected light against a black nonreflecting • Prepare inoculum
surface
o Measure mm

Establishing Diameter Interpretive Breakpoints

• The diffusion test depends on the formation of a
gradient as antimicrobial concentrations as the
antimicrobial agent radially diffuses to the agar.
• The drug concentration decreases as the distance
increases from the disk.
o ↑ distance, ↓drug concentration
• Zone of inhibition is formed at the critical point where
the amount of drug at a specified location in the
medium is unable to inhibit organism growth.

Test Performance
Disk Storage
• Long term storage: -20C or below in a non-frost-free
freezer
• Working supply: 2-8C for at least 1 week
• Disks should always be stored in a tightly sealed
container with desiccant
• Containers should be allowed to warm to room temp
before opening to prevent condensation
• Improper storage indicator: Penicillin and Methicillin
(easily deteriorate)

Inoculation and Incubation

• Plate should be swabbed two or more times turning
the plate 60degrees each time to create an even lawn
• Purity Plate (BAP or CAP) should be inoculated from
the same swab to check purity of isolate
• Plates should not be stacked more than 5 high
• Required temperature may not be obtained located in
the center

E-test (Epsilometer Test)

• Uses the principle of establishing an antimicrobial
density gradient in an agar medium as means of
determining antimicrobial susceptibility
• Uses the plastic test strips impregnated on the
undersurface with an antimicrobial concentration
gradient and marked on the surface with a
concentration index or scale
• MIC is determined where the growth ellipse intersect
the E-test strip
• Low MIC = effective antibiotic

Automated Antimicrobial Susceptibility Test Methods

Principles Used:
• Turbidimetric detection of bacterial growth in a broth
using a photometer
• Detection of hydrolysis of a fluorogenic growth
substrate incorporated in a special test medium
• Matrix-assisted laser desorption/ionization - time of
flight (MALDI-TOF)
o MALDI-MS - determines susceptibility in minutes