Antimicrobial

Susceptibility Testing
Introduction

 AST refers to invitro methods used to determine the

susceptibility of a bacterium to an antimicrobial agent.
 The results assist in determining the most appropriate
antimicrobial agents to treat infections.
 AST is also an important tool to monitor the emergence
and spread of antimicrobial resistance.
Important Points

 Standard inoculum of test bacterium- special suspension

of test bacterium with specified turbidity.
 Turbidity- expressed in the form of Mac Farlands (unit for
expressing turbidity of bacterial suspension)
 Spectrophotometer- used for measuring turbidity of
standard inoculum.
 Temperature for incubation- 35 to 37 degree celcius
 Time of result interpretation- 16 to 18hrs (overnight
incubation)
Antimicrobial Susceptibility Testing
Methods
 AST methods involve culturing a sample to obtain a pure
isolate and testing to determine which antimicrobial
agents inhibit the growth of or kill the pathogen.
 These include:
1. Dilution method (Quantitative)
2. Disc diffusion method (Qualitative)
3. E test (Epsilometer test)
Dilution Method
 Dilution method is the reference method of AST.
1. Agar dilution (solid media)
2. Broth dilution (liquid media)
Microbroth test- petri plates are used
Macrobroth test- test tubes are used

Principle
It is the lowest concentration of antimicrobial agent that will
inhibit the visible growth of microorganism after incubation
Broth dilution
 The medium used in broth dilution is nutrient broth.
 Eg: UTI with E.coli (overnight broth culture is done in
peptone which is used as a standard inoculum.)
 Take same amount of nutrient broth in a series of test
tubes.
 Prepare serial dilution of antibiotics in nutrient broth.
 The test tube with no antibiotic acts as control.
 Add 1ml of standard inoculum in all test tubes
 Incubate overnight at 37 degree celcius
Interpretation
 Control shows maximum growth (maximum turbidity)
 As the concentration of antibiotics increases, turbidity
decreases.
 At a specific concentration where there is no turbidity it
is the MIC (minimum inhibitor concentration).
 MIC is the lowest concentration of the antimicrobial
agent at which there is no visible growth.
 If this whole process is done in petri dish it is called
microbroth dilution.
 Disadvantage- fastidious organisms can not be tested
Agar dilution
 Medium used in agar dilution is Muller Hinton Agar.
 The method involves preparing a series of agar plates
containing antimicrobial agent to be tested in
increasing concentrations.
 Then add standard inoculum and spread it.
 Look for the lowest concentration of antimicrobial agent
that prevents the appearance of colonies.
 Advantage-
fastidious bacteria can be tested.
can be used for anaerobes.
Disc diffusion method
 It is also known as Kirby Bauer method.
Principle
 A paper disk with a defined amount of antibiotic is used
to generate a transparent zone in the agar close to the
disc.
Materials required
• Mueller Hinton Agar
• Actively growing broth or streaked plate of single
organism (pure culture)
• Sterile saline
• Test tube
Materials required

• 5ml pipette
• Swab
• 0.5 McFarland test standard
• McFarland reference card
• Disk dispenser
• Antibiotic disk cartridges
Procedure
 Perform gram staining to confirm culture purity from your
subculture plate.
 Using a sterile 5ml pipette add 5ml of sterile saline to a
sterile test tube
 Using an inoculating loop select several colonies from
your subculture plate and transfer to a tube of sterile
saline.
 Dilute your organism to obtain turbidity equivalent to the
0.5 McFarland test standard.
 Hold your diluted tube and the 0.5 McFarland test
standard against the black lined McFarland reference
card to accurately rate the turbidity.
Procedure
 Within 15 mins of diluting your organism dip a sterile swab
into properly adjusted inoculum. Lift it out of the
suspension and firmly rotate the swab several times
against the upper inside wall of the test tube to
eliminate excess fluid.
 If your swab is too wet your agar surface will not dry
correctly and the antimicrobial agents in the disk will
diffuse through the wet surface and not into the agar.
 Streak the entire surface three times with the swab
turning the plate 60 degrees between streakings (turn
the swab too) to obtain even inoculation.
Procedure
 Close the lid and let it sit for 3-5 mins before applying
drug imprenated disks.
 Apply the disks by means of a dispenser using aseptic
technique.
 Lightly press the disk down with sterile swab to make
contact with the surface.
 Place your agar plate inverted at 37 degree celcius
incubator.
 Procedure
 Streptococcus organisms should be on BHI instead of MHA
plates and incubated with 5-10 percent carbon dioxide.
 Examine the plate after 16-24 hrs incubation.
 Measure (in mm) only zones showing complete inhibition.
 Hold the measuring device over the back of inverted plate
over a black non reflective surface and illuminate from
above.
 Compare the values you obtained with those on disk
disffusion zone diameter chart to determine the susceptibility
level to the antibiotics used.
Report values as
Resistant- indicates that clinical efficacy has not
been reliable in treatment studies.
Intermediate- implies clinical applicability in body
sites where the drug is physiologically concentrated or a
high dosage of drug can be used.
Susceptible- implies that infection due to the
organism may be treated with concentration of
antimicrobial agent used.
E test / Epsilometer test
 It is the combination of disc diffusion and dilution
method.
 The medium used is Mueller Hinton Agar.
 In this method a standard inoculum of 0.5 McF turbidity is
used.
 Instead of disks plastic strips which are impregnated with
graded concentration of antibiotics along its length are
taken.
 The markings along the length of the strip represent the
concentration of antibiotics.
E test / Epsilometer Test
 The concentration at which the zone of inhibition
intersect the plastic strip determine the MIC.