ANTIMICROBIAL SUSCEPTIBILITY TEST

- One of the crucial and sensitive tests.
- 2 Methods
Diffusion Method (Kirby Bauer Techniques)
Dilution Method
- *Note that whenever using the procedure on
diffusion method, should be done as they may
lead to significant differences as a result.
- *Note: Applicable only to aerobic organisms.
No standard procedure for anaerobic
organisms.

Dilution Test Method

Pre-Analytical Phase (Materials)
- Used for determining the minimal
concentrations of antimicrobial agents that will - Broth Culture of Bacteria (TSB)
inhibit or kill the microorganisms.
- In the clinical setting, Normal Saline Solution
- Inoculating the organisms using a dilution of (NSS) is added with a colony, then a streak.
antimicrobial agents incubating at 35°C for 16-
- MHA (4mm in depth)
20hrs.
- Sterile Petri Dish
- MIC is the lowest concentration of the
antimicrobic agent that inhibits the growth of - Filter Paper Disc inpregnated with antibiotics
microorganisms. (Minimal Inhibitory or antibiotic disc
Concentration/MIC)
- Cotton Swabs and forceps
- MBC of antimicrobic is the lowest
- McFarland Standard (Barium Sulfate Turbidity
concentration of antimicrobic agents that kills
Standard)
the microorganisms. (Minimal Bactericidal
Concentration (MBC) *0.5 McFarland Standard

*Used for Standardization of the

inoculum for ASTs
Kirby Bauer Technique (Diffusion
Method) *Purposes
- It is the paper disc method of sensitivity a.Study of bacterial growth curve
testing indicated for the microorganisms in
B. Adjusting the concentration of
which its susceptibility cannot be predicted,
bacterial vaccines
after its identity is known.
C. Research purposes
- At present, the method of choice is a slight
modification of KBT and Turck's method. * 0.5mL of 1% Barium Chloride/ 99.5mL
of 1% Sulfuric Acid
- Kirby Bauer Technique is accepted by US FDA
and US National Committee on Clinical * Dapat same turbidity si McFarland kay
Laboratory Standards subcommittee on Bact. Suspension
Antibiotic Susceptibility Testing.
- Caliper and reading Chart as a template
- Why? Bcos it gives satisfactory results
producing good inter and intra-laboratory
reproducing reports.
Analytical Phase (Steps)
1. Transfer with a wire loop with 3-5 colonies of
organisms to be tested from TSB.
* Dapat pure culture. Similar
morphology same na without flaming
the loop.
Incubate the tube for 2-5hrs.
2. Standardize the inoculum by comparing the
turbidity of the subculture with 0.5 MFTS
against a black background.
3. Adjust the turbidity of subculture.
4. Inoculate the plates by dipping a sterile swab
into the standardized inoculum/subculture.
Remove excess inoculum by pressing and
rotating yhe swab firmly againts the side of the
tube above the level of the liquid. Streak the
swab all over the surface of the medium 3x,
rotating the plate through an angle of 60° after
each application. Finally, pass the swab around
the edge of the agar surface.
5. After the inoculum has dried for 3-5mins,
place the antibiotic discs on the agar by the use
of sterile forceps or by the disc dispenser. A
maximum of 7 discs can be place on a 9-10 cm.
plate. Six discs may be placed in the center of
the plate. Gently press the discs on the agar to
ensure contact.
*Within 30mins of preparation, the
plates should be placed in an incubator
at 35° for 16-18hrs (overnight
incubation).
chart. Zone diameter may be read after
incubation of 6-8hrs if needed. However, the
optimum time is 14hrs. Measurement can be
made on the undersurface of the plate without
opening the lid.
* The endpoint is taken as the
complete inhibition of growth as
determined by the naked eye but there
are 3 exceptions:
1. With Sulfonamide and cotrimoxazole,
slight growth occurs within the
inhibition zone such growth must be
ignored. (??)
2. When B-Lactamase-
producing Staphylococci are tested
against the penicillin, zones of inhibition
are produced within a hip up or clearly
refined edge which are readily
recognisable regardless of ZOI. They
should be reported as resistant.
3. Certain Proteus spp. which swarm
into the zone of inhibition around the
antibiotics. But the zone of inhibition is
usually clear. The swarming growth
should be ignored.
Post-Analytical Phase
- Disinfection, sterilization, and cleaning of the
working area.

* Take note temp above 35°C invalidate

the results for methicillin and oxacillin.

* Do not incubate in an atmosphere

with CO2.

6. Measure zone of diameter (including the

diameter of the disc) using a mm. scale, sliding
Vernier Caliper, template or zone measuring