Antibiotic Antibiotic Sensitivity Sensitivity

TestingTesting

Methods

Technique & Interpretation

Factors affecting

Tukaram Prabhu KAsst ProfessorMicrobiologyPCMS & RC

Lecture taken for II yr MBBS (2008-2009 session)

• The following lecture is compiled from different textbooks & websites and is for teaching - learning use only

• This lecture is not a substitute for classroom lecture / text book but an adjunct for the same

• This lecture may be useful for BSc Nursing, BMLT & DMLT students too

Why is it done?

• To determine the ability of an antibiotic agent to inhibit the in-vitro bacterial growth

• In the laboratory 2 methods are used– Dilution technique

– Diffusion technique

Dilution method

• 2 methods – Agar dilution– Broth dilution

• Done to determine the MIC & MBC– Minimum concentration of antibiotic required to

arrest the multiplication of the bacteria is known as Minimum Inhibitory Concentration

– Minimum concentration of antibiotic required to kill the bacteria is known as Minimum Bactericidal Concentration

MIC = 1/8

MBC = 1/4

Dilution method – when to do?

• Patient does not respond to treatment thought to be adequate

• Relapses while being treated• When there is immunosuppression

Diffusion method

• Disc diffusion most commonly used• Disc of blotting paper impregnated with

known volume and concentration of antibiotic is plate on a lawn culture of test bacteria

• Antibiotic diffuses into the medium• After overnight incubation, the culture is

examined for zones of inhibition

Diffusion method

• Material required – Trypticase soy broth

– Mueller Hinton Agar

– Sterile cotton swab

– 0.5 McFarland tube

– Antibiotic discs

Diffusion method

• Kirby Bauer method– Zone of inhibition produced is compared

against a previously compared standard chart

• Stokes method– Zone of inhibition produced is compared

directly with that of control

Mueller Hinton agar with antibiotics showing zones of inhibition

Kirby-Bauer Chart

Antibiotic S MS R

Amikacin 14 mm 15-16 mm 17 mm

Ciprofloxacin 15 mm 16-20 mm 21 mm

Ceftriaxone 13 mm 14-20 mm 21 mm

Doxycycline 12 mm 13-15 mm 16 mm

Gentamicin 12 mm 13-14 mm 15 mm

Interpretation

• SENSITIVE– A pathogen reported as sensitive suggests

that the infection it has caused is likely to respond to treatment if the antibiotic to which it is susceptible is used in normal recommended doses

Interpretation

• INTERMEDIATE / MODERATELY SENSITIVE– A pathogen reported as MS suggests that

the infection it has caused is likely to respond to treatment if the antibiotic is used in larger than normal doses or when the antibiotic is concentrated at the site of infection

Interpretation

• RESISTANT– A pathogen reported as resistant suggests

that the infection it has caused will not respond to treatment with that antibiotic irrespective of dose or site of infection

Factors affecting AST

1. pH of the environment

2. Components of the medium

3. Stability of the drugs

4. Size of inoculum

5. Length of incubation

6. Metabolic activity of bacteria

7. Depth of the medium

E test

Octodiscs