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AST Practical

Lab

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AST Practical

Lab

Uploaded by

funn forr
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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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

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