Synonyms

⚫ AntibacterialSusceptibility Testing(ABST)
⚫ Antibiotic Sensitivity Testing(AST)

⚫ Antimicrobial Susceptibility Testing

⚫ Antibiogram Test
Antibiotic Sensitivity Testing(AST)
• Antibiotic sensitivity testing, also known as antibiotic
susceptibility testing (AST), determines how effective
a specific antibiotic or antifungal will be against a
bacterial or fungal infection.
• The test is performed by growing bacteria or fungus
from a sample taken from the infected site, and then
exposing the bacteria to different antibiotics.
 OBJECTIVES
⚫ To guide the clinician in selecting the best
antibiotic
• To control the use of inappropriate antibiotics
• To distinguish the range of activity of an antibiotic
• To collect epidemiologic information on
antibacterial resistance
 Types
• For testing of isolates
Qualitative from healthy animals
• Less serious
infections

Quantitative • Critically ill animals

• Serious patients
Disk diffusion methods
⚫ Principle

-Disk impregnated with a defined amount of antibiotic

are placed on agar medium uniformly seeded with the
test organism
⚫A concentration gradient of the antibiotic is formed
by diffusion from the disk into the agar.
⚫ Interact with the freshly seeded test organisms
⚫ If
the organism is killed or inhibited by the
concentration of the antibiotic ,there will be no
growth in the immediate area around the disc:this is
called the

Inhibition zone
edge
Kirby –Bauer Disc Diffusion Method
Materials
Mueller Hinton
Agar

Antibiotic Disks

Turbidity Standard

Swabs
 Mueller –Hinton Agar
⚫ Non-selective, non-differential medium
⚫ Used primarily for the disk diffusion method
⚫ Medium containing beef extract, casein hydrolysate, starch and agar.
⚫ Starch absorb toxins released from bacteria, so that they cannot
interfere with the antibiotics
⚫ It is a loose agar: better diffusion of the antibiotics
⚫ Mueller-Hinton agar is considered best for routine susceptibility
testing
Best Medium – MHA (Mueller Hinton Agar)
1. Shows acceptable batch to batch reproducibility for
susceptibility testing
2. Low in sulphonamide, trimethoprim and tetracycline
inhibitors
3. Gives satisfactory growth of most non fastidious
pathogens, aerobic and facultative anaerobes.
Antibiotic Disks
⚫ Commercially available
⚫ Stocks of antibiotic discs
stored at -140C for 1
month
⚫ Equilibriate with room
temperature before
application
Drugs for routine susceptibility tests
 Set 1: the drugs that are available in most hospitals and
for which routine testing should be carried out for every
strain
 Set 2: the drugs that are tested only:
▪ at the special request of the physician/ veterinarian
▪ or when the causative organism is resistant to the first-
choice drugs
▪ or when other reasons (allergy to a drug, or its
unavailability) make further testing justifiable
Turbidity Standard
0.5 ml solution A
(0.048 M BaCl2)
McFarland
0.5
Turbidity
standard
99.5 ml solution
B
(0.36 N H2 SO4)
 Kirby-Bauer Test
4-5 well isolated colonies are selected

Top of the colonies are touched

Growth transferred to broth

Fresh organism suspended in broth (Bacterial suspension)

Turbidity adjusted by comparison with 0.5 McFarland turbidity

standard

Swab organism all over the plate evenly

 Incubate 370C

Place discs containing specified concentration of different

antibiotics on the plate and incubate

Measure diameter of Inhibition zone

Use tables to assess if zone size indicates resistance or

sensitivity to that antibiotic
Kirby-Bauer Method
Procedure
1.To prepare the inoculum from the primary
culture plate,touch with a loop on the top
of colonies
2.Transfer this growth to a tube of saline
or broth
3.Compare the tube with the 0.5 McFarland turbidity
standard and adjust the turbidity of the test
suspension by adding more bacteria or more saline
4.Inoculate the plates by dipping
a sterile swab into the inoculum
Remove excess inoculum by pressing and
rotating the swab firmly against the side
of the tube above the fluid level
5.Streak the swab all over the the surface in
three directions

6.Finally,pass the swab around the edge of agar surface

7.Leave the inoculated plates to stand for 3-5 minutes for
absorption of excess moisture
8.The antibiotic discs are placed onto agar surface using
-Sterile forceps -Antibiotic disc
dispenser

9.The plates should be placed in an incubator at

35ºC within 15 minutes of preparation
⚫ Each disc is gently pressed to ensure
complete contact with the agar surface
⚫ Centre to centre distance between discs-
24mm
⚫ Distance from the lid edge -15mm

⚫ 6 discs per standard 90mm Petri dish

⚫ Incubated aerobically for 16-18
hours
Measurement of diameter
⚫ Usingautomated zone readers
-BIOMIC
-Vitek
Measuring diameter of zone of
inhibition by using a ruler in
mm
Using a standard table of antibiotic susceptibilities, determine if
the strain is resistant (No zone), intermediate (Small zone), or
susceptible (Larger zone) to the antibiotics tested.
Factors affecting the zone of inhibition
⚫ Size of the inoculum
-Turbidity of medium-0.5 McFarland opacity standard
⚫ Test medium

-Mueller-Hinton agar or its modification

(Isosensitest agar-Oxoid)
⚫ Antimicrobial agent and its concentration in disc
⚫ Incubation conditions

-350c for 16-18 hours under aerobic conditions

⚫ Test bacterium-resistance or susceptibility
⚫ Effects of Variation in Divalent Cations
Results
⚫ Susceptible

⚫ Intremediate

⚫ Resistant
Stokes Method
⚫ Interpretationbased on comparison between zones
seen with the test organism & those of the known
sensitive control.
T > C or T=C Sensitive

T< C  Resistance
Antimicrobial discs
⚫ Storage
⚫ Routine use -Stored at 4oC
⚫ Beta lactam antibiotics-stored for upto one week only
⚫ Long term storage- -140c
⚫ Don’t use expired discs
⚫ Potency of new discs should be checked
Test medium
⚫ Depth of agar
-Mueller –Hinton agar plate depth-4mm
⚫ pH-7.2 and 7.4
⚫ Sterility checking-30-350c for 24hrs
⚫ Storage -4 0 C for 7 days
⚫ Ensure that medium is thymine or thymidine free
 2- Dilution method:
Used to determine the minimal concentration of the antibiotic to inhibit or
kill micro organisms.
Achieved by dilution of antibiotic in either agar or broth media
Achieved by:
1. Tube dilution methods.
2. Agar dilution method.
Minimum inhibitory concentration (MIC): The lowest concentration of
antibiotic that inhibit growth of bacteria.
Highest dilution of an antibiotic required to inhibit the growth of a
bacterium
Minimum bactericidal concentration (MBC): Lowest
concentration of antibiotic that kills bacteria isolated from
patient.
The minimum bactericidal concentration (MBC) can be
obtained by sub culturing from each tube (showing no growth)
onto a nutrient agar plate without any antimicrobial agent The
tube containing the lowest concentration of the drug that fails to
show growth, on subculture, is the MBC of the drug for that test
Strain.
Tube / Broth dilution
⚫ Organism is added to tubes containing decreasing amounts of
the antibiotic
⚫ Tubes are prepared by adding two fold dilutions of antibiotic in
a series
⚫ Each tube is inoculated with a suspension of test bacterium

⚫ Incubation at 35-370C for 24 hrs

⚫ Lowest concentration of drug that inhibits growth is the MIC
Two types of dilution methods can be used:
a) Micro-dilution method:
 It is performed in 96 well microtiter plate.
 We use about 0.1 ml total broth volume.
b) Macro-dilution method
We use test tubes for this test.
We use about 1 ml total broth volume in which we
make 2-folds dilution of antibiotic.
Agar dilution/Agar incorporation tests
⚫ Serialdilutions of the drug are prepared in agar
and poured into plates
Advantage
• Many strains can be inoculated on each
plate containing an antibiotic dilution
• It directly measures the MBC; there is no need
of sub culturing as it is done with broth
dilution method.
 E-Test
⚫ Epsilometer test
⚫ Quantitative method of antibiotic sensitivity testing

⚫ Applies both dilution of antibiotic and diffusion of

antibiotic into the medium
⚫ Combines the principles of disk diffusion and agar
dilution methods

Dilution

E-Test
Diffusion
PROCEDURE
⚫ Apply E-test strip on an inoculated agar plate
⚫ Immediate release of drug

⚫ Incubation of plate

⚫ Symmetrical inhibition of ellipse is produced

⚫ The intersection of the inhibitory zone edge and the

calibrated carrier strip indicates the MIC with
inherent precision and accuracy
AST of Fastidious organisms
⚫ Most fastidious organisms do not grow well enough
in routine antibiotic testing systems and require
some type of supplementation
⚫ 5-10% sheep blood agar -Streptococci or
Actinomyces pyogenes
Application of Computers in Antibacterial
Susceptibility Testing
⚫ ‘WHONET’- Software developed for the
management of routine laboratory results by WHO

⚫ Useful in supplying current guidelines, protocols to

local laboratories, in identifying the clusters of
resistant isolates and emerging outbreaks, research
studies.
References
⚫ Quinn PJ, Carter ME, Markey B & Carter GR. 1994.
Clinical Veterinary Microbiology. Wolfe Publ. pp-95-102
⚫ NCCLS Manual of Antimicrobial Susceptibility Testing
THANK
YOU..