1. Kirby-Bauer disk diffusion Testing skill based learning
Dr.T.V.Rao MD
Dr.T.V.Rao MD

2. Principles of susceptibility testing
Dr.T.V.Rao MD

3. Kirby-Bauer Test is a agar disk diffusion method
Kirby – Bauer Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant bacterial isolates.
Dr.T.V.Rao MD

4. Kirby-Bauer antibiotic testing
Kirby-Bauer antibiotic testing (KB testing or disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated paper disks to test whether particular bacteria are susceptible to specific antibiotics. A known quantity of bacteria are grown on agar plates in the presence of thin filter paper discs containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of inhibition).
Dr.T.V.Rao MD

5. Agar disk diffusion method
Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.4
Antibiotic storage oC minimum
disks temperature
Inoculum McFarland 0.5 (108 bacteria/mL)
Incubator temperature 35oC
atmosphere ambient air 5 5

6. Procedure
Prepare a pure culture (18-24 hrs) of the sample on a non- selective medium
Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standard
Use a Wickerham Card as background when adjusting the turbidity
Sample
0.5 McFarland Standard
Dr.T.V.Rao MD

7. Aseptic transfer of colonies for propagation of bacteria
Select at least 4 to 5 well-isolated colonies of the same morphological type from an agar plate. Touch the top of each colony with a wire loop and transfer the growth to a tube containing 4 to 5 mL of a suitable broth medium, such as tryptic-soy broth. Allow the broth culture to incubate at 35°C until it achieves or exceeds the turbidity of 0.5 McFarland standard.
Dr.T.V.Rao MD

8. Choosing a colony for testing
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9. McFarland 0.5 and Adjusted Test Organism
Dr.T.V.Rao MD

10. Inoculate the plate with uniformity
Within 15 minutes after adjusting the turbidity of the inoculum suspension, dip a sterile non-toxic swab on an applicator into the adjusted suspension. Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab.
Dr.T.V.Rao MD

11. Uniform inoculum on plates is essential for effective detection
Inoculate the dried surface of a Muller-Hinton agar plate by streaking the swab over the entire sterile agar surface. Repeat this procedure two more times, and rotate the plate 60° each time to ensure an even distribution of inoculum. Replace the plate top and allow 3 to 5 minutes, but no longer than 15 minutes, for any excess surface moisture to be absorbed before applying the antibiotic disks.
Dr.T.V.Rao MD

12. Procedure
Within 15 minutes of adjusting the turbidity
dip a sterile cotton swab into the sample
streak a lawn of bacteria on Mueller-Hinton agar
Leave the lid agar for 3-5 minutes (no more than 15 minutes) to allow plate to dry
Dr.T.V.Rao MD

13. Be cautious
The agar medium should have pH 7.2 to 7.4 at room temperature.  The surface should be moist but without droplet of moisture. The antibiotic disks should be maintained at 8°C or lower or freeze at -14°C or below until needed, according to the manufacturer's recom­ mendations. Allow the disks to warm to room temperature before use. Don't use expired disks.
Dr.T.V.Rao MD

14. Getting ideal results depend on right inoculum
There should be an almost confluent lawn of growth when done properly. If only isolated colonies grow, the inoculum was too light and the test should be repeated. To avoid extremes in inoculum density, never use undiluted overnight broth cultures for streaking plates./li>
Dr.T.V.Rao MD

15. Procedure Apply antibiotic impregnated disks on the bacterial lawn
Important: where the disk drops is where it stays
Incubate for hours at 33 ± 2oC unless otherwise instructed
Dr.T.V.Rao MD

16. Do not load the plates with too many antibiotic disks
Place the appropriate disks evenly (no closer than 24 mm from center to center) on the surface of the agar plate either by using a sterile forceps or the dispensing apparatus. No more than 12 disks should be placed on one 150 mm plate or more than 5 disks on a 100 mm plate. A disk is not to be moved once it has come in contact with the agar surface since some of the compound diffuses almost instan­taneously.
Dr.T.V.Rao MD

17. Proceed with incubation of plates
Invert the plate and place them in an incubator at 35°C within 15 minutes after disks are applied. The plates should be incubated aerobically . After hrs. of incubation, examine each plate and measure the diameters of the zones of complete   inhibition,   including   the diameter of the disk. Measure the zones to the nearest millimeter using a ruler. Large colonies growing within a clear zone of inhibition should be subcultured, reidentified and retested to know whether they are contaminants or mutants
Dr.T.V.Rao MD

18. Interpretation c and C are the minor and major breakpoints
The main concept is the “clinical categorisation"
Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints
c and C are the minor and major breakpoints
Susceptible
Intermediate
Resistant
MIC < c
≤ MIC < C
≤ MIC

19. Understanding breakpoints
Words of laboratory specialists
It is not possible to work alone
Breakpoints are the expression of a consensus among the scientific community at a given time in a country
Breakpoints are determined using two approaches
Pharmacological concept
Epidemiological concept

20. Results Antibiotics diffuse out onto the agar
Concentration of antibiotics decrease as they diffuse further away from the disks
After incubation, observe for a clearing on the bacterial lawn (zone of inhibition)
Bacterial growth
incubation
Zone of inhibition
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21. Follow the guidelines for measuring zones of resistance and susceptibility
Interpret the zone sizes by referring to the manufacturer provided standard table and report the organism to be  either susceptible, intermediate, or resistant. Never compare the zone sizes of two different antibiotics and judge their effectiveness accordingly.
Dr.T.V.Rao MD

22. Results Measure the diameters of the zone of inhibition
Interpret the results as “resistant” or “susceptible” according to the guideline provided by the CLSI
Interpretation of the zone of inhibition is different for each bacteria-antibiotic combination
Dr.T.V.Rao MD

23. Disk Susceptibility Testing Problems proper inoculum and heavy inoculum
Dr.T.V.Rao MD 23 23

24. Disk Susceptibility Testing Problems disk properly applied disk not properly applied
Dr.T.V.Rao MD

25. An agar gel that is too thick leads to smaller zones
Dr.T.V.Rao MD

26. Standard strains for quality assurance
Precision and accuracy ensured through control strains
Known susceptibility to antimicrobial agents
Standard strains include
Staphylococcus aureus ATCC 25923
Escherichia coli ATCC
Pseudomonas aeruginosa ATCC 27853

27. E-test® Same principle as the Kirby-Bauer Test
Uses a plastic strip with a predefined gradient of antibiotic concentration
Results are read directly on the strip where the zone of inhibition intersects with the strip E
Zone of inhibition
Bacterial growth
Dr.T.V.Rao MD

28. Results
Interpret results as “resistant” or “susceptible” according to the guidelines provided in the package insert
For ambiguous results, refer to the provided reading guide for :
Organism related effects
Drug related effects
Resistance mechanism related effects
Technical and handling effects
Dr.T.V.Rao MD

29. Patient results may be incorrect if:
The organism was misidentified
A clerical error was made
Inappropriate choice of antimicrobials were tested and reported
The wrong patient’s sample was examined
The wrong test was ordered
The sample was not preserved properly

30. Antimicrobial Susceptibility Testing Goals
When possible, don’t duplicate systems
Meet regulatory and other guiding requirements
Use automation when possible and technically sound
Have flexibility in antimicrobial battery
Focus technologist time on tasks that utilize their training and expertise
Get accurate results out as soon as possible
Large CF population
Disc diffusion quality control testing
65% agents concurrently being tested for quality assurance on existing Etest™ batteries
Advances in Vitek allow for Dtest, cefoxiting and VRSA screen to be automated
DO NOT COMPROMISE QUALITY FOR SPEED

31. Programme created by Dr. T. V
Programme created by Dr.T.V.Rao MD for Microbiologists in the Developing World
Dr.T.V.Rao MD