This document discusses antibiotic sensitivity testing (AST) to determine the most suitable antibiotic against a microorganism. It describes direct and indirect susceptibility testing methods and the common disk diffusion method. The disk diffusion method involves placing antibiotic disks on an inoculated agar plate and measuring inhibition zones to interpret if a microorganism is susceptible, intermediate, or resistant. Mueller-Hinton agar is typically used as the growth medium and factors influencing inhibition zone size are discussed. The E-test method and dilution methods for quantitative antibiotic susceptibility testing are also summarized.

1. Antibiotic Sensitivity Testing (AST)
9th Practical Lab
Yousif H.M.Sharif
MSc Biomedical Sciences
Hull University/GB

2. Learning objectives
A. Mechanism of antibiotic resistant
B. Understand standard antimicrobial susceptibility testing
C. Interpret antimicrobial susceptibility testing

3. Antimicrobial resistant: Results from
misuse, overuse, under/ inadequate use of
antimicrobials
 Costs money, lives and undermines
effectiveness of health delivery
programs
 Threat to global stability and national
security

4. AS a guide for treatment
• Antimicrobial sensitivity testing : to determine the most suitable
antibiotic agent against specific microorganism (drug of choice)
.WHY?
• Widespread and non selective antibiotics usage results in a high
driving force in the development of antibiotic resistances
As an epidemiological tool
• The emergence of resistant strains of major pathogens (e. g.
Shigellae, Salmonella typhi, Mycobacterium tuberculosis)
• Continued surveillance of the susceptibility pattern of the
prevalent strains (e. g. Staphylococci, Mycobacterium tuberculosis,
Gram-negative bacilli)
Susceptibility test, main purposes

5. Direct versus indirect susceptibility tests
Indirect method
• cultured plate from pure culture .
Direct method
• Pathological specimen • e.g. urine, a positive blood culture, or a
swab of pus
Colony pick up adjust turbidity spread the inoculum

6. Common methods used for AST
1. Agar diffusion based methods (qualitative method)
• Disc diffusion method (also called Kirby-Bauer method)
• Epsilometer test (E-test)
2.Dilution method (Quantitative method) :Rare used

7. Principle
The Kirby-Bauer (K-B) is the most common method for antibiotic
resistance/susceptibility testing. The results of such testing help
physicians in choosing which antibiotics to use when treating a sick
patient
Test utilizes small filter disks impregnated with a known
concentration of antibiotic. The disks are placed on a Mueller-Hinton
agar plate that is inoculated with the test microorganism.
Upon incubation, antibiotic diffuses from the disk into the
surrounding agar. If susceptible to the antibiotic, the test organism
will be unable to grow in the area immediately surrounding the disk,
displaying a zone of inhibition .

8. Mueller-Hinton agar (MHA)
MHA is the best medium to use for routine susceptibility testing
using Kirby-Bauer disc diffusion method for nonfastidious bacteria
(both aerobe and facultative anaerobe). Use of media other than
Mueller-Hinton agar may result in erroneous results.
Formula for Mueller-Hinton agar per liter of purified water
• Beef extract: 2.0 g
• Acid Hydrolysate of Casein: 17.5 g
• Starch: 1.5 g
• Agar: 17.0 g
Placing antibiotic disc in Mueller-Hinton
agar medium

9. Why mueller-hinton agar is used in routine
antibiotic susceptibility testing (AST) ?
• It shows acceptable batch-to-batch reproducibility for
susceptibility testing
• It supports satisfactory growth of most nonfastidious pathogens
• It is low in sulfonamide, trimethoprim, and tetracycline inhibitors
(i.e. concentration of inhibitors thymidine and thymine is low in
MHA)
• A large body of data and experience has been collected
concerning susceptibility tests performed with this medium.

10. Modifications of Muller Hinton agar
• Mueller Hinton agar medium supplemented with 5% sheep
blood is recommended for determining the antimicrobial
susceptibility of
– Streptococcus pneumoniae
– Neisseria meningitidis
The disk diffusion test. Virulent Streptococcus pneumoniae on the surface
of Mueller-Hinton agar with 5% horse blood. Susceptible to all tested
antibiotics.

11. Procedure of K-B methods
1.Clinical specimens e.g urine
2.Isolation and identification e.g E.coli
• Note bacterial infections are usually caused by one pathogenic
bacteria except anaerobic infections which are mixed infection
3.Preparation of inoculum

12. 3. Preparation of inoculum
• Using a sterile inoculating loop, touch four or five isolated colonies
of the organism to be tested.
• Suspend the organism in 2 ml of sterile saline.
• Vortex the saline tube to create a smooth suspension.
• Adjust the turbidity of this suspension to a 0.5 McFarland
standard by adding more organism if the suspension is too light.
• Use this suspension within 15 minutes of preparation.

13. In microbiology, McFarland standards are used as a reference to adjust
the turbidity of bacterial suspensions, if the bacterial suspension is too
turbid, it can be diluted with more diluent. If the suspension is not
turbid enough, more bacteria can be added
McFarland Nephelometer Standards

14. 4. Dip a sterile cotton swab into bacterial suspension, after mixing
it then remove the swab and excess fluid is expressed by pressing
and rotating the swab against the inner sides of the tube above
the level of the liquid.

15. 5-Streak the swab all over the surface of the sensitivity testing
medium (Mueller-Hinton agar) three times in different directions,
rotating the plate through an angle of 60º after each application .
Finally, pass the swab around the edge of the agar surface, by this
you get a lawn growth. Discard the swab in disinfectant, leave the
plate to dry a few minutes at room temperature with the lid closed.
(the depth of the medium in Petri dish 4mm→25 ml of the
medium).

16. 6-Pick up an antimicrobial disc with the sterile forceps and place it on the
surface of the agar medium, each disc should be gently pressed down to
ensure even contact with the medium, using the tips of the forceps. Six
discs can be placed on 90 mm plate .five discs may be spaced evenly about
15 mm from the edge of the plate and one disc in the center . Also can be
used antibiotic disc dispenser to apply discs on surface of the medium.

17. 7- Invert the plates and incubate at 37Cº for 18 to 24 hours.
After incubation the diameter of each inhibition zone (including the
diameter of the disc) should be measured and recorded in mm. the
measurements can be made with the ruler from the back (under
surface) of the plate with out opening the lid. The diameter of each
inhibition zone is compared to the standard charts .

19. Inhibition zone:-
The area where no bacteria grow
after overnight incubation

20. Interpretation and Reporting of the Results
• Using the published EUCAST guidelines, determine the
susceptibility or resistance of the organism to each drug tested
(Table 1).
• For each drug, indicate on the recording sheet whether the zone
size is susceptible (S), intermediate (I), or resistant (R) based on
the interpretation chart.
• The results of the Kirby-Bauer disk diffusion susceptibility test are
reported only as susceptible, intermediate, or resistant. Zone
sizes are not reported to physicians.

22. Depend on the diameter of inhibition zone ,the bacteria
divided to :-
Sensitive S →it means the bacterium responds to the
antimicrobial agent.
Intermediate I →it means the bacterium is not fully
sensitive to the antimicrobial agent.
Resistant R →it means the bacterium is not effected by the
antimicrobial agent.

23. What factors influence the size of the zone
of inhibition ?
1. Pathogen susceptibility
2. Antibiotic diffusion effects
3. Agar depth : 4mm is good ,to avoid false susceptible or resistant
4. pH: 7.2 to 7.4 pH
5. Size of the inoculated organism:
6. Presence of other metals :Excessive thymidine or thymine can
reverse the inhibitory effects of sulfonamides and trimethoprim
resulting in smaller and fewer distinct zones of inhibition, or no
zones at all.
7. the temperature and length of incubation
Microorganisms that are resistant to an antibiotic will not show a
zone of inhibition (growing right up to the disk itself) or display a
relatively small zone.

24. E-TEST
The E test (also known as the Gradinet Diffusion Method) is based
on the same principle as the disk diffusion method.
It is an in vitro method for quantitative antimicrobial susceptibility
testing
E-Test determines the Minimum Inhibitory Concentration (MIC).
Following incubation, the MIC value (ug/ml) is read at the point of
intersection between the zone edge and the E-Test strip.

25. Materials
• E-test strips (store frozen)
• Mueller Hinton Agar (MH)
Procedures
• The same suspension of the test organism in sterile saline
equivalent to a 0.5 McFarland standard using isolated colonies
will be used as in the previous experiment (disk method).
• Using a sterile cotton swab, inoculate the organism onto an
appropriate agar plate, streaking in 3 directions over the entire
agar surface.
• Using needle, apply the Imipenem antimicrobial strip onto the
agar.
• Incubate plate at 35oC x 20 to 24 hours

26. Interpretation OF E.Test
• After incubation read the MIC value at the point of intersection
between the zone edge and the E-test strip. Since E-test
comprises a continuous gradient, MIC values in between two-fold
dilutions can be obtained. Always round up these values to the
next two-fold dilution before interpretation.

30. Dilution tests (Quantitative):
This test is used to determine the Minimum Inhibitory Concentration
(MIC) of antimicrobial agent and Minimum Bactericidal
Concentration(MBC)
e,g. Broth (Tube) dilution method :-
This test can be done by adding the same volume of standardized
inoculum to a series of tubes containing two fold dilution in nutrient
broth of the antimicrobial being test. After overnight incubation at
37Cº the tube that shows no growth (no turbidity) with lowest
concentration of drug represents MIC. Also MBC can be determined
by inoculating loopfuls from the tubes not showing growth into
suitable agar medium .
The plate showing no growth represents MBC.
MIC:-(Minimum Inhibitory Concentration)
The lowest concentration of antimicrobial agent that inhibits
bacterial growth after overnight incubation (no turbidity) .
MBC :-(Minimum Bactericidal Concentration)
The lowest concentration of antimicrobial agent that kills or prevents
bacterial growth after subculture of bacteria to a media.

33. Any Questions?
Thank You