The presentation about the principles, procedures, and result interpretation of antibiotic sensitivity test

1. Antibiotic sensitivity tests
Prepared by Dr Mohammed Iraqi
Assistant researcher at animal Health Research Institute (AHRI), GIZA,
Egypt

2. Definition
• A test done to check the effectiveness of a drug against a
bacterium and to select the best drug that acts against
the bacterium.
• The in vitro testing of bacterial cultures with antibiotics
to determine susceptibilityofbacteriatoantibiotictherapy

4. Why Need
continues for
testing
for Antibiotic
Sensitivity?
• Bacteria can develop resistance
following repeated or subclinical
(insufficient) doses, so more advance
antibiotics and synthetic
antimicrobials
are continually required to overcome
them.

5. Qualitative Methods Quantitative Methods

6. • Diffusion methods:
• Principle:
• A paper disk with a defined amount of
antibiotic is used to generate a
dynamically changing gradient of
antibiotic concentrations in the
agar in the vicinity of the disk.

8. • The antibiotic contained in a reservoir diffuses out of the disk to form
the gradient.
• The test organism starts to divide and grow and progresses toward a
critical mass of cells.
• Inhibition zone is formed at the critical time where a particular
concentration of the antibiotic is just able to inhibit the organism
before it reaches an overwhelming cell mass.

11. Mueller-
Hinton-Agar:
• Medium containing beef infusion,
peptone, and starch.
• Used primarily for the disk-diffusion
method.

12. Antibiotic disks:
• Any commercially available discs
with the proper diameter and
potency can be used.
• Stocks of antibiotic discs can be
stored at -20 °C for 1 month.
• On removal from the refrigerator,
the containers should be left at
room temperature for about 1 hour
to allow the temperature to
equilibrate.

13. Turbidity standards:
• Standard inoculum should have turbidity equivalent to 0.5 McFarland
standard.
• Should be from a freshly overnight growth.
• These are made by dissolving barium sulphate in water with
different concentration.
• 0.5 McFaraland have a turbidity equivalent to 1x 105

14. Cotton swabs

15. Procedures
Application o f Antibiotic Discs
Incubation
At 35⁰C for 16 -18 hours
Measurement of inhibition zone diameter

16. 1- To prepare the inoculum from the primary culture plate, touch with
the loop the tops of 3-5 colonies of similar appearance of the organism
to be tested.

17. 2-Transfer this growth to a tube of saline or MHB
(Mueller-Hinton-Broth) and incubate the tube in 37ʗ
for (0.5- 2 hours).

18. 3- Compare the tube to adjust with the
turbidity standard by adding more bacteria or
more sterile saline.

20. the swab all over the surface
of the
medium three times, rotating the plate
through an angle of 60⁰ after each
application.
Finally, pass the swab round the edge
of the agar surface.

21. 7-Leave the inoculum to for a few
minutes at room temperature with the lid
closed.
8-The antibiotic discs may be placed on the
inoculated plates using
• sterile forceps.
• a template.
• a sterile needle tips.
• antibiotic disc dispenser.

23. • A maximum of can be placed on a 9–10 cm plate.
• Six discs may be spaced evenly, approximately 15 mm from
the edge of the plate, and 1 disc placed in the center of the
plate.
• The plates should be incubated within 30 minutes of
preparation.

24. Strips of multiple antibiotics can be placed in one go

25. Measurement of inhibition zone
diameter
• Using a ruler on the under-surface of the
plate containing transparent medium.
• Using a pair of calipers on the plate
containing opaque medium.

26. Measurement of diameter
using automated zone readers

27. Interpretation of
results:(according
to standard zone
diameter
detected by CLSI
(Clinical and
Laboratory
Standard Institute
Interpretative chart of zone
sizes
Diameter of zone
inhibition (mm)
Antibiotic Resistant Intermediate Susceptible
Tetracycline <14 15-18 >19
Chloramphenicol <12 13-17 >18
Cotrimoxazole <10 11-15 >16
_
Erythromycin <13 14-22
Gentamycin <12 13-14

28. Stokes
method

30. Dilution method:
• Used to determine the
minimal concentration of
antibiotic to inhibit or kill the
microorganism.
• Achieved by dilution of
antibiotic in either agar or
broth media.

31. Minimum inhibitory concentration (MIC):

34. 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

36. Interpretation of E-test

37. USES OF E-test:
•Detecting
– Glycopeptide-resistant Enterococci (GRE)
– Glycopeptide-intermediate S. aureus (GISA)
– Resistant Mycobacterium tuberculosis – Extended spectrum
beta lactamases (ESBL)