ANTIBIOTIC SENSITIVITY TESTING
DEFINITIONS
Minimum Inhibitory Concentration (MIC)
• Lowest concentration of antimicrobial agent that prevents in-vitro growth of bacteria
• Antimicrobial concentration is expressed in microgram/millilitre
Minimum Bactericidal Concentration (MBC)
• Lowest concentration of the drug that kills the bacteria.
• Done by sub-culturing from the tubes
Antibiotic sensitivity tests are of 2 types:
• Diffusion tests
• Dilution tests
Diffusion Test
• Here, the drug is allowed to diffuse through a solid media so that gradient is
established
• the concentration is highest near the site of application of the drug and decreases with
distance.
• The test bacterium is seeded on the medium and its sensitivity to the drug is
determined from inhibition of its growth.
• Several methods have been used for application of drug
• The most commonly used method is to use filter paper discs impregnated with
antibiotics
Disc Diffusion Test
• Kirby Bauer Method is commonly used
• It uses filter paper discs, 6 mm in diameter charged with appropriate concentration of
the drugs
• The discs are stored dry, in the cold.
• They may be prepared in the laboratory or purchased commercially
• A suitable dilution of a broth culture or a broth suspension of the test bacterium if
blooded on the surface of a solid medium (Mueller Hinton agar or Nutrient agar)
• Inoculation may also be performed by spreading with swabs
• After drying the place (37OC for 30 mins), antibiotic discs (6 per 9 cm plate) are
applied with sterile forceps
• After overnight incubation, the degree of sensitivity is determined by measuring the
zones of inhibition of growth around the discs
• Growth will be inhibited around the discs containing antibiotics to which the
bacterium is susceptible
• But growth will not be inhibited around those antibiotics to which the bacterium is
resistant
• The diameter of the zone is influenced by a variety of factors such as
• Diffusibility of drug
• Disc concentration
• Nature and composition of medium
• Thickness of media
• Presence of inhibitory or stimulatory substances
• pH
• time of inoculation
• The results are reported as
• Sensitive
• Intermediate
• Resistant
• To the different drugs
EXAMPLE FOR KIRBY BAUERS DISK DIFFUSION TEST – 1
EXAMPLE FOR KIRBY BAUERS DISK DIFFUSION TEST – 2
EXAMPLE FOR KIRBY BAUERS DISK DIFFUSION TEST – 3
Dilution Tests
• Here, serial dilutions of the drug are prepared and inoculated with the test bacterium
• Dilution tests are generally employed when
• the therapeutic dose is to be regulated accurately as in the treatment of
bacterial endocarditis
• for tests on slow growing bacteria such as tubercle bacilli
• and when small degrees of resistance are to be demonstrated
• It is too laborious for routine use
• Dilution tests maybe done by
• Tube dilution method
• Agar dilution method
Tube Dilution Method
• Macro-dilution
• It is done using test tubes
• Micro-dilution
• It is done using microtube wells that are moulded into plastic plates
• Advantages
• Small volume of reagents is used
• Large no. of bacteria can be tested
Epsilometer test (E-test)
• Quantitative method of detecting MIC by using principle of both dilution and
diffusion
• The anti-microbial content of the strip is graded and concentration is printed linearly
along the strip
• MIC is read from the point on the strip where the ‘zone of growth’ inhibition passes
Interpretation of Antibiotic Susceptibility Test
Organism: Staphylococcus aureus
Antibiotic susceptibility Test: Kirby-Bauer Disk Diffusion Method
Zone CLSI M100 2023
S.
Antibiotic size Interpretation Susceptible Intermediate Resistant
No.
(mm) (≥ mm) (mm) (≤ mm)
1 Cefoxitin 22 - 21
2 Erythromycin 23 14-22 13
3 Clindamycin 21 15-20 14
4 Gentamicin 15 13-14 12
5 Linezolid 21 - 20
6 Ciprofloxacin 21 16-20 15
Interpretation of Antibiotic Susceptibility Test
Organism: Escherichia coli
Antibiotic susceptibility Test: Kirby-Bauer Disk Diffusion Method
Zone CLSI M100 2023
S.
Antibiotic size Interpretation Susceptible Intermediate Resistant
No.
(mm) (≥ mm) (mm) (≤ mm)
1 Amikacin 20 17-19 16
2 Ceftriaxone 26 23-25 22
3 Cefixime 19 16-18 15
4 Cefotaxime 23 20-22 19
5 Ofloxacin 16 11-15 12
6 Levofloxacin 21 17-20 16
7 Nitrofurantoin 17 15-16 14
Interpretation of Antibiotic Susceptibility Test
Organism: Pseudomonas aeruginosa
Antibiotic susceptibility Test: Kirby-Bauer Disk Diffusion Method
Zone CLSI M100 2023
S.
Antibiotic size Interpretation Susceptible Intermediate Resistant
No.
(mm) (≥ mm) (mm) (≤ mm)
1 Amikacin 17 15-16 14
2 Imipenem 19 16-18 15
3 Ceftazidime 18 15-17 14
4 Ofloxacin 16 13-15 12
5 Piperacillin 22 18-21 17
6 Piperacillin
22 18-21 17
Tazobactam
