This document provides information on antibiotic sensitivity testing methods. It discusses the key terms related to antibiotic sensitivity like bacteriostatic, bactericidal, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). It then describes the two main types of antibiotic sensitivity tests - diffusion tests like Kirby-Bauer disk diffusion method and dilution tests like broth dilution method. For diffusion tests, it explains how to prepare media, inoculum, antibiotic discs and controls and interpret the results. It also discusses Epsilometer or E-test method to detect MIC. For dilution tests, it details the broth dilution method to determine MIC and MBC.

1. ANTIBIOTIC SENSITIVITY TEST

2. Antibiotic
• An antibiotic is a substance produced by various species of
living microorganisms.
• (e.g. bacteria and fungi)
Terminology:
Bacteriostatic:
Bactericidal:
Minimum inhibitory concentration:
• MIC is the smallest amount of an agent needed to inhibit growth
of a microorganism.
Minimum bactericidal concentration:
• MBC is the smallest amount of an agent needed to kill the
microorganism.

3. INTRODUCTION
• Pathogenic bacteria shows great strain wise
variations in susceptibility to antimicrobial agents.
• It is, therefore, essential to determine the
susceptibility of isolates to antibiotics that are likely
to be used in treatment.
• AST is performed only for pathogenic bacteria
isolated from the specimens and not for the
commensals.

4. ANTIMICROBIAL SENSITIVITY TESTS
Two types:
Diffusion tests-
• Strokes disc diffusion method
• Kirby-Bauer disc diffusion method
Dilution tests-
• Broth dilution method
• Agar dilution method

5. A. DIFFUSION TESTS
• Disc diffusion tests are most widely used to
determine the susceptibility of isolates of pathogenic
bacteria to antibiotics that are likely to be used in the
treatment.
• Here, the antibiotic is allowed to diffuse through the
solid medium so that concentration is highest near
the site of application of the antibiotic disc and
decreases with the distance.
• These tests is not suitable for slow growing
microbes.

6. • In the disc diffusion method uses filter paper discs
charged with appropriate concentration of the drugs.
• The test bacterium is inoculated on the medium and
these antibiotic disc are applied.
• Sensitivity to the drug is determined from the
inhibition of bacterial growth around the disc.

7. Medium:
• The medium should support a good overnight
growth of the test and control organisms.
• Mueller-Hinton agar may be used for the testing
aerobes and facultative anaerobes.
• Nutrient agar is another alternative medium used.
• The medium is prepared in petridish (100 mm in
diameter).
• The depth (thickness) of the medium should be 3-4
mm (around 25ml of the medium is used).
• The plates may be stored at 4oC for up to one week.
• pH - 7.2 to 7.4.

8. • A more acidic pH decreases the activity of
aminoglycosides and macrolide antibiotics such as
erythromycin.
• A more alkaline pH favours the action of
tetracycline , novobiocin and fusidic acid.
• Some drug such as sulphonamides and
trimethoprim, are inhibited by thymidine present in
culture medium .
• 5% lysed horse blood is added to the medium to
neutralise the inhibitory effect of thymidine.
• The addition of 5% Nacl to the medium is required
for detecting resistance to methicillin in strains of
Staphylococci.

9. INOCULUM
• The organisms are isolated in pure culture on a solid medium.
• Isolated colonies are inoculated in a suitable broth medium
and incubated at 35-37oC for 4-6 hours.
• The density of the organisms in broth is adjusted to
approximately1.5X108 cfu/ml by comparing its turbidity with
that of 0.5 Mc farland opacity standard tube.
• This broth is inoculated on the medium by spreading with
sterile swabs.
• The ideal inoculum after overnight incubation gives even
semi-confluent growth.
• To heavy an inoculum reduces the size of inhibition zones.
• Similarly, inoculum of control strain should also be prepared
as in the case of test strain.

10. Control Strains
• Control strains for Stokes and Kirby - Bauer disc
diffusion methods are shown below-
Test bacteria Control strain
Stokes Kirby-bauer
Coliforms Esch. coli NCTC 10418 Esch. Coli ATCC 25922
Pseudomonas P. Aeruginosa NCTC 10662 P. Aeruginosa ATCC 27853
Enterococci E.Faecalis NCTC 12697 E.Faecalis ATCC 29212
Haemophilus spp. H. Influenzae NCTC 11931 H. Influenzae ATCC 49247
N. gonorrhoeae N. gonorrhoeae (sensitive strain ) N. gonorrhoeae ATCC
49226
Other organism that can
grow aerobically
Staph. Aureus NCTC 6571 Staph. Aureus ATCC 25923

11. ANTIBIOTIC DISCS
• Antibiotic discs may be prepared in the
laboratory or purchased commercially.
• Filter paper discs of 6 mm in diameter are used.
• For preparation of disc in laboratory, pure salt of
antimicrobial agents should be used and not those
use for clinical practice.
• Distilled water serve to dissolve most antibiotic
powders but some antibiotics may require
different diluents:-

12. Antibiotic Diluent
Chloramphenicol Ethanol
Rifampicin Ethanol
Erythromycin Ethanol
Nitrofurantoin NaOH solution
Sulphonamides NaOH solution
Trimethoprim Acetic acid
Amoxycillin NaHCO3 (Saturated)
ceftazidime NaHCO3 (Saturated)
Table: Diluent of various antibiotics

13. • Disc concentration of antimicrobial agent are
shown in table:-
Antibiotics Disc concentration
Benzyl penicillin 1.2 µg
Ampicillin 10 µg
Amoxicillin 20 µg
Cephalexin 30 µg
Methicillin 5 µg
Carbenicillin 100 µg
Gentamycin 10 µg
Amikacin 30 µg
Erythromycin 15 µg
Tetracycline 30 µg
Chloramphenicol 30 µg
Nalidixic acid 30 µg
Trimethaprim 5 µg
Ciprofloxacin 1 µg

14. • The disc are stored at low temperature.
• Hence, disc should be taken out from refrigerator 1-
2 hours before applying on the medium.

15. 1.Stokes Disc Diffusion Method
• The test bacterium is inoculated on the central one
third and control on upper and lower thirds of the
plate.
• However, in Modified Stokes Disc Diffusion Method,
the test bacterium is inoculated over the upper and
lower thirds of the plate and control on central one
third.
• Antibiotic disc are applied between the standard and
test inocula.
• A maximum of six antibiotic discs can be applied on
a 100 mm diameter plate.
• The plates are then incubated at 37oC for 16-18 hrs.

16. Reporting:
• Plates in which growth is not semi confluent should not be
reported unless the test is repeated.
• The reporting of results is done by comparing the zones of
inhibition of control and test bacterium.
• Measure the zone of size i.e. the distance in mm from edge
of the disc to the zone edge.
Interpretation:-
Sensitive:
• The zone of test bacterium is equal to, or larger than that of
control strain.
• If zone size of the test bacterium is smaller than that of
control, the difference between two should not be at least 3
mm

17. Intermediate sensitive:
• The zone size of the test bacterium should be
at least 2 mm and the difference between the
zone of test and control strain should be at
least 3 mm.
Resistant:
• The zone size of the test bacterium is smaller
than 2 mm.

18. Stokes disc diffusion method and modified stokes disc
diffusion method

19. 2. KIRBY-BAUER DISC DIFFUSION METHOD
• In this method cotton swab dip into the inoculum.
• Inoculate the Mueller- Hinton agar plate by
streaking the swab three times over the entire agar
surface.
• Allow 3-5 minute for the surface of agar to dry
before applying the antibiotic discs, by using 100
mm diameter petridish, seven disc may be applied,
one in centre and six in periphery.
• The plates are then incubated at 37oC for 16-18 hrs.

20. • The zones of complete growth inhibition around
each of the disc is included in this measured.
• The diameter of disc is included in this
measurement.
• The interpretation of zone size into sensitive,
intermediate or resistant is based on interpretation
chart.
• Control strains of Staph. aureus , Esch. Coli, Ps.
aeruginosa etc. should be tested each time when a
new batch of agar or discs is used.

21. Kirby- Bauer Disc Diffusion Method

22. • Interpretation Chart Used In Kirby – Bauer Disc
Diffusion Method
Antibiotic Zone size in mm
S I R
Ampicillin(AMP) 17 ─ 16
Chloramphenicol (C) 18 13-17 12
Ciprofloxacin (CIP) 21 16-20 15
Doxycycline (DO) 16 13-15 12
Fosfomycin (FO) 16 13-15 12
High level Gentamycin (HLG) 10 7-9 6
High level Streptomycin (HLS) 10 7-9 6
Levofloxacin (LE) 17 14-16 13
Linezolid (LZ) 23 21-22 20
Nitrofurantion (NIT) 17 15-16 14
Norfloxacin (NX) 17 13-16 12
Not for UTI Erythromycin 23 14-22 13
Penicillin (PN) 15 ─ 14
Teicoplanin (TEI) 14 11-13 10
Tetracycline (TE) 19 15-18 14
Vancomycin (VA) 17 15-16 14
Zone Diameter for Enterococcus spp.

23. Antibiotic Zone size in mm
S I R
Amikacin (AK) 17 15-16 14
Azithromycin (AZM) 18 14-17 13
Cefoxitin (CX) for CONS 25 ─ 24
Cefoxitin (CX)for S.aureus ≥ 22 ─ ≤ 21
Co- Trimoxazole (COT) 16 11-15 10
Doxycycline (DO) 16 13-15 12
Gentamycin (GEN) 15 13-14 12
Linozolid (LZ) 21 ─ 20
Netilmycin (NET) 15 13-14 12
Nitrofrunction (NIT) 17 15-16 14
Norfloxacin (NX) 17 13-16 12
Not for UTI Erythromycin(E)
Clindamycin (CD)
23 14-22 13
Penicillin (P) ≥ 29 ─ ≤ 28
Sulfonamides (SF) 17 13-16 12
Teicoplanin (TEI) 14 11-13 10
Tetracyclin (TE) 19 15-18 14
Tobramycin (TOB) 15 13-14 12
Trimethoprim (TR) 16 11-15 10
Zone Diameter for Staphylococcus spp.

24. Antibiotic Zone size in mm
S I R
Amikacin (AK) 17 15-16 14
Aztreonam (AZT) 22 16-21 15
Cefexime (CXM) 18 15-17 14
Ceftazidime (CAZ) 18 15-17 14
Ciprofloxacin (CIP) 21 16-20 15
Colistin (CL) 11 ─ 10
Gentamycin (GEN) 15 13-14 12
Jmipenem (IMP) 19 16-18 15
Levofloxacin (LE) 17 14-16 13
Meropenem (MRP) 19 16-18 15
Netilmycin (NET) 15 13-14 12
Piperacillin (PI) 21 15-20 14
Piperacillin(PI)-Tazobactum 21 15-20 14
Polymyxin B (PB) 12 ─ 11
Tobramycin (TOB) 15 13-14 12
Zone Diameter for Pseudomonas (NLF, Oxidase Positive)

25. I Line Drugs
Antibiotic Zone size in mm
S I R
Amikacin (AK) 17 15-16 14
Amoxycillin(AMP) - Clavulanate 18 14-17 13
Ampicillin (AMP) 17 14-16 13
Ampicillin(AMP) - Sulbactum 15 12-14 11
Cefotaxime (CTX) 26 23-25 22
Cefoxitin (CX) 18 15-17 14
Ceftazidime (CAZ) 21 18-20 17
Ceftriaxone (CTR) 22 20-22 19
Ciprofloxacin (CIP)for other enterobacteriaceae 21 16-20 15
Co-Trimoxazole (COT) 16 11-15 10
Gentamycin (GEN) 15 13-14 12
Tobramycin (TOB) 15 13-14 12
II Line Drugs
Aztreonam (AZT) 21 18-20 17
Cefepime (CPM) 25 ─ 18
Chloramphenicol (C) 18 13-17 12
Doxycyclin (DO) 14 11-13 10
Imipenem (IMP) 23 20-22 19
Levofloxacin (LE) 17 14-16 13
Meropenem (MRP) 23 20-22 19
Nalidixic Acid 19 14-18 13
Netilmycin (NET) 15 13-14 12
Piperacillin (P) 21 18-20 17
Piperacillin- Tazobactum (PIT) 21 18-20 17
Zone Diameter for Enterobacteriaceae

26. PRIMARY DISC DIFFUSION TEST
• The disc diffusion methods, as described
above, are done after the pathogenic bacteria
are isolated from the clinical specimens.
• When results are required urgently, the
‘primary disc diffusion test’ may be performed.
• Here, the clinical specimen is directly
inoculated uniformly on the surface of a plate
and the antibiotic discs are applied.
• The results of the primary test should be
verified by testing the isolates subsequently.

27. Epsilometer or E-test
• E-test is antimicrobial sensitivity test to detect
minimum inhibitory concentration (MIC) of
antibiotic.
• It is recent modification of agar diffusion test.
• It uses an absorbent strip with a known gradient of
antibiotic concentration along its length.
• When the strip is placed on the agar plate
inoculated with the test organism, the antibiotic
diffuse into the medium.
• The minimum inhibitory concentration (MIC) is
recorded as the lowest concentration of the
gradient which inhibits the growth of the organism.

28. E- Test Or Epsilometer On The Agar Plate
MIC

29. B. DILUTION TESTS
1.Broth dilution method:
• Serial dilution of the drug in Mueller-Hinton broth
are taken in tubes and a standardised suspension of
the test bacterium inoculated.
• An organism of known sensitivity should also be
titrated.
• Incubate at 37oC for 16-18 hrs.
• For determination of MIC of methicillin incubate at
30oC.
• The minimum inhibitory concentration (MIC) is
ready by noting the lowest concentration of the drug
at which there is no visible growth.

30. • The minimum bactericidal concentration (MBC) is
determined by subculturing from each tube showing
no growth on nutrient agar plate without any
antimicrobial agent.
• Incubate the plates and examine them for growth.
• The tube containing the lowest concentration of the
drug that fails to show growth, on sub culture, is the
MBC of the drug for that test strain.

31. Uses of MIC determination:
• When the therapeutic dose is to be regulated
accurately as in the treatment of bacterial
endocarditis.
• For testing antimicrobial sensitivities of slow
growing bacteria such tubercle bacilli.
• When small degree of resistance are to be
demonstrated.

32. Antibiotic
concentration
in (/ml)
Mueller Hinton
broth
No Growth Turbidity (growth)
No Growth Growth
MIC=8µg /ml
MBC=32µg /ml
Broth Dilution Method Showing MIC And MBC

33. 2.Agar dilution method:
• Serial dilutions of the drug are prepared in agar
and poured into plates.
• Many strains can be inoculated on each plate
containing an antibiotic dilution.
• This method is more convenient when several
strains are to be tested at the same time.

34. Automated antimicrobial susceptibility
tests
• Several automated systems are available now,
such as,
 VITEK 2 bacterial identification and
antimicrobial sensitivity system (bioMerieux)
 Phoenix System