Laboratory 2

Antimicrobial Susceptibility Testing

 Antimicrobial Susceptibility Tests
• Minimal inhibitory concentration [MIC]
– The smallest concentration (or highest dilution)of
antibiotic that inhibits the growth of an organism.

• Minimal bactericidal concentration [MBC]

– The smallest antibiotic concentration showing
99.9% killing of bacteria.
 Antimicrobial Susceptibility Tests
• Liquid media (dilution)
– Microdilution: testing volume 0.05 – 0.1 mL
– Macrodilution: testing volume >1.0 mL

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• • 96 well microtiter plate
 Antimicrobial Susceptibility Tests
• Solid media (diffusion)
– Disk diffusion (Kirby-Bauer)
– E-tests
 Antimicrobial Susceptibility Tests
• Goal: To determine whether organism
expresses resistances to agents potentially
used for therapy
• Standardization
National Committee for Clinical Laboratory
Standards (NCCLS)

Name changed to:

Clinical Laboratory Standards Institute

(CLSI)
 Broth Dilution
• Inoculum size:

– 0.5 McFarland 10⁸ CFU/ml

– 1: 100 dilution 10⁶ CFU/ml
– 1:2 dilution 5 x 10⁵ CFU/ml
 Broth Dilution
• Prepare:
– 9 empty tubes
– Muller-Hinton broth
– Drug solution
– Bacterial culture (10⁶ CFU/ml)
– Graduated pipettes
 Broth Dilution
1. Add 1 ml MHB to tubes 1-9
2. Add 1 ml drug solution to tube 1
3. Mix well the content in tube 1, then transfer 1 ml to tube 2
4. Repeat step 3, till tube 8
5. Discard 1 ml from tube 8
6. Add 1 ml culture into tubes 1-9
7. Incubate at 37°c for 18-24 hours
8. Look for the MIC (μg/ml)
9. Transfer 0.1 ml from non-turbid tubes into agar plates and spread
10. Incubate at 37°c for 18-24 hours
11. Look for MBC
0.5 ml from nonturbid tubes were added to 2 ml of MHB.
From this dilution, 0.2 ml were taken
and sub cultured to agar and incubated overnight.

2 x 10⁴ CFU were present in the 0.2 ml that was inoculated

on the agar plates. Therefore, the inoculation from the
clear MIC tubes that showed an agar plate count of 20
colonies or less was considered as the MBC
Disk Diffusion
Test
 Disk Diffusion Technique (Kirby-Bauer)
• Principle:
– A Petri-plate containing an agar
medium is inoculated with a standard
amount of organism.
– Next, filter paper disks impregnated
with a known quantity of
chemotherapeutic agents are placed on
the agar plate where the drugs would
diffuse from disks into the agar.
– If effective, a zone of inhibition is
formed around the disk.
 Prepare inoculum
suspension

Select colonies
 Mix well

Standardize inoculum
suspension
 Swab plate

Remove sample
 Incubate overnight

Add disks
Measure Zones
 Disk Diffusion Test

• Qualitative results
– Susceptible
– Intermediate
– Resistant
 Standardization

– Inoculum size

– Growth medium

– Incubation atmosphere, temperature, duration

– Antimicrobial concentrations used

 Standardization

Inoculum preparation
− 0.5 McFarland
− Adjust by using turbidity meter
 Standardization
Growth medium
– Mueller-Hinton agar
– Depth of agar: 4 mm
– pH of agar: 7.3

An agar gel that is too thick leads to

smaller zones
Modify methods for
fastidious bacteria
Standardization
Standard strains for quality assurance
• Precision and accuracy ensured through control strains
– Known susceptibility to antimicrobial agents

• Standard strains include

– Staplylococcus aureus ATCC 25923
– Escherichia coli ATCC 25922
– Pseudomonas aeruginosa ATCC 27853
E test Read MIC
where elipse
intersects
scale
• Uses the principle of
predefined antibiotic
gradient on a plastic strip
to generate an MIC value
• Processed like the disk
diffusion
• Individual antibiotic strips
are placed on an
inoculated agar surface
• After incubation, the MIC
is read where the
growth/inhibition edge Read plates
intersects the strip after
recommended
Incubation
Reading E-tests Ciprofloxacin for
Yersinia pestis

Resistant > 4 ug/ml

Intermediate 1-4 ug/ml

Susceptible < 1

Upper reading