This document discusses drug susceptibility testing, which determines if microbes like bacteria or fungi can be inhibited by antimicrobial drugs. There are two main types of testing - qualitative for less serious infections, and quantitative for serious infections. The Kirby-Bauer disk diffusion method is commonly used, involving placing disks with antibiotics on agar plated with a test organism. The size of the inhibition zone is measured to determine resistance or susceptibility. Factors like incubation time and temperature affect results. Minimum inhibitory concentration methods like broth dilution are also used but are more complex. Computer programs help analyze and track susceptibility data.

1. DRUG SUSCEPTIBILITY
TESTING
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2. INTRODUCTION
 Drug Susceptibility- Susceptibility is a term used when microbe such
as bacteria and fungi are unable to grow in the presence of one or
more antimicrobial drugs.
 Susceptibility testing is performed on bacteria or fungi causing an
individual’s infection after they have been recovered in a culture of
the specimen.
 Bacteria and fungi have the potential to develop resistance to
antibiotics and antifungal drugs at any time.
 This means that antibiotics once used to kill or inhibit their growth
may no longer be effective.

6. OBJECTIVE OF ANTIBIOTIC SUSCEPTIBILITY TESTING

7. TYPES
 QUALITATIVE
 For testing of isolates from “healthy” patients with intact immune defences.
 For less serious infections such as uncomplicated urinary tract
infections.
 QUANTITATIVE
 In the treatment of serious infections such as endocarditis or
osteomyelitis.
 For infections in high risk patients groups such as
immunocompromised patients (eg. Transplant patients)

10. Disk diffusion method
 Principle
• Disk impregnated with a defined
amount of antibiotic are placed on
agar medium uniformly seeded
with the test organism.

11. KIRBY-BAUER DISC DIFFUSION METHOD
 . Material required
Mueller Hinton agar
Antibiotic disc
Turbidity standard
swabs

12. MUELLER HINTON AGAR
 Non selective, Non- differential medium
 Used primarily for the disk diffusion method
 Medium containing beef extract casein hydrolysate, starch, and agar.
 Starch absorb toxin released from bacteria, so that they cannot
interfere with the antibiotics.
 It is a loose agar: better diffusion of the antibiotics.
Robust red agar(Solieria robusta)
Source of agar

13. ANTIBIOTIC DISC
 Commercially available
 Stocks of antibiotic disc stored at -
14° C for 1 month
 Equilibriate with room temperature
before application

14. Turbidity Standard
0.5 ml solution A
(0.048 M BaCl2)
99.5 ml solution B
(0.36 N H2SO4)
 Mc Farland
 0.5
 Turbidity
standard

15. KIRBY-BAUER test / sold agar test
1. Fresh organism suspended in
broth
2. Swab organism all over the
plate evenly
3. Place disc containing
specified concentration of
different antibiotics on the plate

16. .
4. Incubate at 37°C
5. Measure diameter of Inhibition
zone
6. Use tables to assess if zone size
indicates resistance or sensitivity
to that antibiotic.

17. PROCEDURE OF KIRBY-BAUER TEST
1. TO PREPARE THE INOCULUM FROM THE
PRIMARY CULTURE PLATE, TOUCH WITH A LOOP
ON THE TOP OF COLONIES

18. 2. TRANSFER THIS GROWTH TO A TUBE OF
SALINE OR BROTH

19. 3. COMPARE THE TUBE WITH THE 0.5 MAC
FARLAND TURBIDITY STANDARD AND
ADJUST THA TURBIDITY OF THE TEST
SUSPENSION BY ADDING MORE BACTERIA
OR MORE SALINE

20. 4. INOCULATE THE PLATE BY DIPPING A
STERILE SWAB INTO THE INOCULUM
 Remove excess inoculum by
pressing and rotating the swab
firmly against the side of the
tube above the fluid level.

21. 5. STREAK THE SWAB ALL
OVER THE SURFACE IN THREE
DIRECTION.
6. FINALLY PASS THE SWAB
AROUND THE EDGE OF AGAR
SURFACE
7. LEAVE THE INOCULATED PLATES TO STAND
FOR 3-5 MINUTES FOR ABSORPTION OF EXCESS
MOISTURE.

22. 8. THE ANTIBIOTIC DISC ARE PLACED ONTO AGAR
SURFACE USING -
 STERILE FORECEPS • AUTOMATED DISC DISPENSER

23. • Each disc is gently pressed to ensure complete
contact with the agar surface
• Centre to centre distance between disc 24mm
• 6 disc per standard 90mm petri disc.
9. THE PLATES SHOULD BE PLACED IN AN
INCUBATOR AT 35°C WITHIN 15 MINUTES OF
PREPARATION.
• Incubated aerobically for 16-18 hours

24. MEASUREMENT OF INHIBITION ZONE
DIAMETER
 USING RULER
 USING A PAIR OF CALLIPERS.
 TRANSPARENT MEDIUM FROM
THE BACK OF THE PLATE
 OPAQUE MEDIUM OVER THE
SURFACE OF AGAR

26. FACTORS AFFECTING THE ZONE OF
INHIBITION
 Size of the inoculum
o Turbidity of medium -0.5 Mc Farland
opacity standard
 Test medium
o Mueller Hinton agar on its modification
(isosensitest agar, oxoid)
o Its has good batch to batch reproducibility
o Low in sulphonamide, trimethoprim and
tetracycline inhibitors
o Satisfactory growth of pathogen.
 Antimicrobial agent and its
concentration in disc
 Incubation conditions
o 35C for 16-18h under
aerobic conditions
 Test bacterium – resistance or
susceptibility
 Effects of variation in divalent
cations.

27. Results
Results of the testing are usually reported as:
 Susceptible — likely, but not guaranteed to inhibit the pathogenic
microbe; may be an appropriate choice for treatment
 Intermediate — may be effective at a higher dosage, or more
frequent dosage, or effective only in specific body sites where the
antibiotic penetrates to provide adequate concentrations
 Resistant — not effective at inhibiting the growth of the organism in
a laboratory test; may not be an appropriate choice for treatment

29. STOKES METHOD
 Interpretation based on comparison between zones seen
with test organism & those of the known sensitive control.
• Only followed in certain European
countries.
• On the same plate- antibiotic disc, control
strain and test strain placed.
• Incubated at same conditions
• Therefore, no need of any tables to
compare
• Advantage – easy to do
• Disadvantage- MIC cannot be determined
T = zone of inhibition of test
organism
C= zone of inhibition of control
organism

31. DILUTION METHOD (REFERENCE METHOD)
 AGAR DILUTION (Solid media)
 BROTH DILUTION (Liquid media)
o Microbroth test (petri plates)
o Macrobroth test (test tubes)

32. BROTH DILUTION METHOD
 MEDIUM- NUTRIENT BROTH
 E.g. UTI Escherichia coli overnight broth
culture in peptone standard inoculum
 In macrobroth dilution method ,we will take
same amount of nutrient broth in series of test
tubes
 Serial dilution of antibiotics prepared in nutrient
broth control with no antibiotics
 Add 1 ml of standard inoculum to all test
tubes Incubate overnight at 37°C

33. • Control – maximum growth (maximum turbidity)
• As the concentration of antibiotics increases, turbidity decreases
• At a specific conc. – no turbidity – minimum inhibitor concentration
(MIC)
• MIC- lowest concentration of drug at which there is no visible growth.
• If the whole process is done in petri dish- microbroth dilution
• Disadvantage -cumbersome procedure
-Fastidious organisms cannot ne tested.

34. AGAR DILUTION (Solid media)
 Medium – cation adjusted Muller Hinton agar
Prepare serial dilution
Add standard inoculum
Spread it
Look for lowest concentration of
prevent the appearance of colonies.
Advantage – fastidious bacteria can be tested
- can be used for anaerobes
Disadvantage- cumbersome procedure.

35. E-TEST/ EPSILOMETER TEST
 Combination of dilution and disc diffusion
method
 MEDIUM - Muller Hinton Agar
 STANDARD solution of 0.5 mc farland turbidity
 Instead of discs, plating strips impregnated
with graded concentration of antibiotics (serial
dilution) along its length

36. • The concentration at which the
zone of inhibition intersect the
plastic will determine the mic
Advantage- Easy to do,
quantitative method
Disadvantage- Sometimes
results may confuse- go for
dilution or disc diffusion

37. APPLICATION OF COMPUTERS IN
ANTIBACTERIAL SUSCEPTIBILITY
TESTING
 WHONET – software developed for the management of
routine laboratory results by WHO.
 Useful in supplying current guidelines, protocols to local
laboratories, in identifying the clusters of resistant isolates and
emerging outbreaks, research studies.

38. BIBLIOGRAPHY
 Street, T. (Updated 2014 March 13). Antimicrobial Susceptibility.
Medscape. Available online at
https://emedicine.medscape.com/article/2103786-overview?
 L.Barth Reller, Melvin Weinstein, James H. Jorgensen, Mary Jane
Ferraro ANTIMICROBIAL SUSCEPTIBILITY TESTING: A review of
general principle and contemporary practices. (Updated 2009
December 01) Issue 11, Volume 49.()PG. 11749-1755
 IMAGES SOURCE
 Reseachgate.net