Antimicrobial susceptibility testing (AST) determines the resistance of microorganisms to antimicrobial agents. There are several methods for AST including disk diffusion, dilution methods, and E tests. Guidelines for AST are provided by CLSI and EUCAST. Mueller Hinton agar is the recommended medium for testing and inoculum preparation follows the 0.5 McFarland standard. Quality control using standard reference strains is important to ensure accurate and reproducible AST results.

1. Antimicrobial Susceptibility
Testing
By,
Dr.Malathi.M
II year PG, M.D., Microbiology
Chengalpattu medical college

2. Introduction
• Once an organism is isolated , characterization
frequently includes tests to detect antimicrobial
resistance, which is the prime important key
component for the physician.
• The procedures used to produce the
antimicrobial susceptibility profiles and detect
resistance to therapeutic agents are referred to as
antimicrobial susceptibility testing (AST)
methods.

3. TESTING METHODS:
• Methods that directly measure the activity of
one or more antimicrobial agents against a
bacterial isolate
• Methods that directly detect the presence of a
specific resistance mechanism in a bacterial
isolate
• Special methods that measure complex
antimicrobial organism interactions

4. GUIDELINES
• CLSI 2014 (CURRENT)
• EUCAST

5. Media
DiscInoculum

6. MEDIA IN AST
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
Media

7. • Store the plates at 2 to 8 deg C
• Use within 7 days of preparation
• Each batch of MHA plates should be checked for
sterility control
• MHA added with 2% NaCl
• MHA added with defibrinated sheep blood 5%s

8. Factors affecting AST
• pH
• Moisture
• Effects of Thymidine/thymine
• Divalent cations

9. Antibiotic Stock solutions
• Buy commercial pure source of antibiotics
• Don’t use injectable solutions
• Accurate weighing of powders is must
• Standard strains of stock cultures should be
used to evaluate the stock solution
• After preparing the stock solution, make 5 ml
aliquots and frozen it
Disc

10. Calculation of stock solution
1000
--------- * V * C = W
P

11. DRIED FILTER PAPER DISCS:
• Whatmann no.1 filter paper is made to form a
disc size of 6mm
• Keep in petridish and sterilize in a hot air oven
• With the help of antibiotic delivery loop which
has a 20G wire with diameter of 2mm  the
antibiotic is delivered (0.005ml)

12. Storage of discs
• Refrigerate at 2 to 8 deg C
• Beta lactam class drugs should be frozen
• The drugs should be kept outside at RT 1 to 2
hours before work
• The dispensing apparatus which is used to
deliver the drugs should also be refrigerated
• Check the expiry of drugs

13. Inoculum – Standard:
• O.5 Mcfarland standard
• Prepared by 0.5ml of 0.048mol/L BaCl2 and
99.5ml of 0.18mol/L of H2SO4
• Added with constant stirring
• Turbidity standard is checked by
spectrophotometer – 625nm – absorbance
should be 0.008 to 0.10
Inoculum

14. • Seal the tubes containing the Mcfarland
standards and store in dark at RT
• The standard turbidity should be mixed
throroughly every time before use
• Check the density monthly and replace it
monthly

15. AST  METHODS
• Disk diffusion method
• MIC method
• E Test
• Automated systems

17. CONVENTIONAL AST
DISK DIFFUSION METHOD:
• Simplest and most convenient
• Widely used everywhere
• Developed by kirby, sherrris, bauer and turk in
1966
• Types:
1. Kirby Bauer method
2. Stokes method

18. KIRBY BAUER METHOD
• Types:
1. Direct colony suspension method
2. Inoculum (log phase ) method

19. Application of Discs
• 150 mm plate  12 discs
• 100 mm plate  6 discs
• Drug should not be relocated
• Distance from the lid edge  15mm
• Distance between two drug from center to
center  24mm
• Inoculum  disc placement  incubation (
only 15 minutes delay is acceptable each)

20. Kirby baeur disc method

21. INTERPRETATION:
• After 16 to 18 hours
• Confluent lawn of growth
• Zones of inhibition  uniformly circular
• Read with reflected light
• For MRSA  read in transmitted light
• When proteus is tested  thin veil of
swarming growth after the zone of inhibition
should be ignored

22. STOKES METHOD
• Built in controls against many variables and
provide dependable results
• A standard sensitive strain of the bacterium is
inoculated in the middle third of the culture plate
• Standard strains:
• S.aureus ATCC 25923
• E.coli ATCC 25922
• P.aeruginosa ATCC 27853

23. Stoke method

24. • The test bacterium is inoculated in the upper
and lower third of the plate
• Antibiotic discs are placed between the
standard and test inocula so that zones of
inhibition formed around each disc are
composed of standard and test bacteria.
• The results are reported as Susceptible,
Intermediate susceptible, Resistant

25. DILUTION METHODS
• The minimum concentration of antimicrobial
to inhibit or to kill the microorganism is
determined
• MIC
• Broth dilution method
• Agar dilution method

26. • MIC – lowest concentration of antimicrobial
that will inhibit the visible growth of an
organism in an ideal growth condition
• MBC – least concentration of the test antiiotic
which will completely kill the bacteria tested

27. BROTH DILUTION METHOD
• Media: cation adjusted MH broth with a pH of
7.2 to 7.4
• For Haem.influenzae – HTM
• For TMP-SMX – Thymidine free medium
• For oxacillin Resistance – MH broth with 2%
NaCl

28. Working antibiotic solution
• Take original stock solution
• Prepare stock dilutions of the antibiotic of
concentrations 1000 and 100ug/ml
• Arrange two rows of 12 sterile 7.5*1.3cm
capped tubes in the rack
• Take a 30ml universal screw capped bottle
• Add 8ml broth and required antibiotic
concentration and mix the contents

29. • Take 2ml + 2ml and put it in first tube in both
rows
• Remaining 4ml broth  add 4ml fresh broth
• Transfer 2ml+2ml and put in second tube
• Continue this dilution upto 11 tubes
• 12th tube  control

30. INOCULATION
• Inoculation of 1st row with one drop of overnight
broth culture 1 in 1000 dilutions
• 2nd row with control with known sensitivity
organisms
• Final inoculum = 5 * 105 cfu/ml
• Incubate at 37degC for 16 to 18 hours
• Inoculate another tube with 2ml broth and keep
at 4degC in a refrigerator overnight to be used as
standard for determinattion of complete
inhibition

31. Broth dilution method

32. INTERPRETATION
• Positive control tube – turbidity
• Negative control tube – clear
• MIC end point is read
• Special comments:
1. For TMP-SMX
2. Trailing

33. MICROBROTH DILUTION METHOD
• Use double strength mueller hinton broth
• 4 X strength antibiotic solutions prepared as
serial 2 fold dilutions
• Test organism is 2X106 /ml
• Done in 96 well plate

34. AGAR DILUTION METHOD
• 1ml concentration of the drug + 24 ml of MHA
• Inoculum – make 1:10 dilution of
0.5Mcfarland standard inoculum which
delivers 107 cfu/ml
• Using pipette or calibrated loop , deliver 0.001
ml on the surface of the agar giving the final
inoculum of 104 cfu /spot
• Inoculate a control plate

35. Agar dilution method

36. INTERPRETATION
• Examine the drug free control growth of the
test organism for viability and purity
• Place the plate on a dark background and
examine them for the lowest concentration
that inhibits visible growth
• A single colony of a faint haze is not recorded
as growth

37. E test
• Epsilometer test
• An exponential gradient testing methodology
• A predefined stable antimicrobial gradient is
present on a thin inert carrier strip
• Following incubation, the E strip releases drug
and a symmetrical inhibition ellipse is
produced
• MIC = intersection of the inhibitory zone edge
and the calibrated carrier strip

38. E test

39. QUALITY CONTROL IN AST
• QC: a process in the laboratory designed to
monitor the analytical phase of testing
procedures to ensure that tests are working
properly
• CLSI recommends use of ATCC strains for QC in
AST

40. • QC strains should be included daily with the
test ideally
• Not more than 1 in 20 results should be
outside the accuracy limits
• No zone should be more than 4 SD away from
midpoint between the stated limits

41. • Due to expense, it can be switched as once
weekly testing
• Perform QC for 30 days and with less than
10% inaccuracy , continue for weekly
• Test repeated for each new drug included
• All documentation maintained indefinitely

42. REFERENCE STRAINS FOR QC
• Beta lactamase negative – ATCC E.coli 25922
• Beta lactamase positive – ATCC E.coli 35218
• Aminoglycosides – P.aeruginosa ATCC 27853
• Thymidine levels – E.feacalis ATCC 29212
• Cephalosporins – H.influenzae ATCC 49766
• Beta lactamase negative – ATCC staph.25923
• Beta lactamase positive – ATCC staph 38591

43. REFERENCES
• Diagnostic Microbiology – Bailey and Scott
13th edition
• CLSI guidelines 2014
• Quality control in AST – ijmm journal
• Practical microbiology – Mackie and
Mccartney – 14th edition
• Image courtesy – google search engine