Antibacterial Susceptibility Test

INTRODUCTION:
Antimicrobial susceptibility testing is important in production of new drugs, epidemiology,

and prediction of therapeutic outcome (Balouiri et al., 2015). Detection of possible drug
resistance in common pathogens and assurance of susceptibility to certain drugs for a particular
infection are the main goals for testing. Susceptibility testing of individual isolates is important
with species that may possess acquired resistance mechanisms such as members of
Enterobacteriaceae, Pseudomonas species, Staphylococcus species, Enteroccocus species,
and Streptococcus pneumonia (Barth, et al., 2009). Modern medicine depends on
chemotherapeutic agent such as antibiotic to treat infectious disease by killing pathogenic
microorganism (bacteriocidal) or inhibit their growth (bacteriostatic) at concentrations low
enough to avoid undesirable damage to the host. Determination of antimicrobial effectiveness
against specific pathogens is essential for proper therapy. Testing can show which agents are
most effective against pathogen.
For this reason, discovery of new antibiotics is an exclusively important objective.
Natural products are still one of the major sources of new drug molecules today. They are
derived from prokarypticbacteris, eukaryotic microorganisms, plants and various animal
organisms. Microbial and plant products occupy the major part of the antimicrobial compounds
discovered until now (Balouiri et al., 2015).
One method that is used to determine antibiotic susceptibility is the sensitivity disk
method of KirbyBauer. In this method, antibiotics are impregnated onto paper disks and then
placed on a seeded Mueller-Hinton agar plate using a mechanical dispenser or sterile forceps.
The plate is then incubated for 16 to 18 hours, and the diameter of the zone of inhibition around
the disk is measured to the nearest millimeter. The inhibition zone diameter that is produced will
indicate the susceptibility or resistance of a bacterium to a specific antibiotic. Antibiotic
susceptibility patterns are called antibiograms can be determined by comparing the zone
diameter obtained with the known zone diameter size for susceptibility (Harley-Prescott:
Laboratory Exercises in Microbiology, 5th Edition, 2002).
Balouiri, M., Sadiki, M., and Ibnsouda S.K. Methods for in vitro evaluating antimicrobial activity:
A review. Journal of Pharmaceutical Analysis 6 (2016) 71-79.
https://doi.org/10.1016/j.jpha.2015.11.005
L. Barth Reller, Melvin Weinstein, James H. Jorgensen, Mary Jane Ferraro, Antimicrobial
Susceptibility Testing: A Review of General Principles and Contemporary Practices, Clinical
Infectious Diseases, Volume 49, Issue 11, 1 December 2009, Pages 1749–
1755, https://doi.org/10.1086/647952
Harley, J.P. and Prescott, L.M. (2002) Laboratory Exercises in Microbiology. 5th Edition, The
McGraw-Hill Companies.
WORKSHOP PROPER
KIRBY –BAUER DISK DIFFUSION SUSCEPTIBILITY TEST
EXPECTED LEARNING OUTCOMES:

At the end of the training, you should be able to:
1. Demonstrate and explain the concept of antibiotics.
2. Discuss the general methodology and the principle in the use of disk diffusion type of
assay for antibiotic activity.
MATERIALS:
A. Culture Media
Mueller Hinton Agar (MHA)
B. Bacterial Culture
Pseudomonas aeruginosa
Escherichia coli
Staphylococcus aureus
C.Reagents
Sterile saline solution
D. Glassware
Screw capped tubes
Test tubes
Petri plates
E. Equipment
Incubator
F. Others:
0.5 McFarland standard
Wickerham card
Caliper or foot rule
Antibiotic disks
Forceps
Antibiotic disk dispenser
Inoculating loop
Bunsen burner
PROCEDURES:
A. MHA PREPARATION
1. Suspend the MHA in 1 liter of distilled water. Mix thoroughly.
2. Heat the solution with frequent agitation and boil for 1 minute to completely dissolve the
components.
3. Autoclave at 121 °C for 15 minutes.
4. Dispense the MHA to to Petri plates to a depth of 4mm (approximately 25 ml of liquid
agar for 100 mm plates and 60 ml of liquid agar for 150 mm plates) and allow to solidify
at room temperature.
5. pH of the MHA should fall between 7.2 and 7.4 at room temperature after solidification.
6. Appropriately label each MHA plate for each organism to be tested.
B. MCFARLAND STANDARD
1. Add a 0.5 aliquot of a 0.048 mol/liter BaCl2 to 99.5 ml of 0.18 mol/liter H2SO4 with
constant stirring to maintain suspension.
2. Verify the correct density of the turbidity standard by measuring absorbance using
spectrophotomer with a 1cm light path and matched cuvette. The absorbance at 625 nm
should be 0.08 to 0.13 for 0.5 McFarland standard.
3. Transfer the barium sulfate suspension in 4 to 6 ml aliquots into screw-cap tubes of the
same size as those used in standardizing the bacterial inoculums.
4. Tight seal the tubes and store in the dark at room temperature.
5. As you would introduce bacterial colonies to the saline in the “preparation of inoculum”,
you should compare the resulting suspension to the McFarland standard. This is done
by holding both the standard and the inoculums tube side by side and no more than 1
inch form the face of the Wickerham card and comparing the appearance of the lines
through both suspensions. If the bacterial suspension appears lighter than the 0.5
McFarland standard, more organisms should be added to the tube from the culture plate.
If the suspension appears to be denser than the 0.5 McFarland standard, additional
saline should be added to the inoculums tube in order to dilute the suspension to the
appropriate density (Figure 1).
C. INOCULUM PREPARATION
1. Using a sterile inoculating loop or needle, touch four or five isolated colonies of the
organism to be tested.
2. Suspend the organism in 2 ml sterile solution.
3. Vortex the saline tube to create a smooth suspension
4. Adjust the turbidity of this suspension to a 0.5 McFarland standard by adding more
organism of the suspension is too light or diluting with sterile saline if the suspension is
too heavy.
5. Use this suspension within 15 minutes of preparation.
D. INOCULATION OF BACTERIAL SAMPLES INTO THE MH PLATE

1. Dip a sterile swab into the inoculum tube.
2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure to
remove the excess fluid. The swab should not be dripping wet (Figure 2).
3. Inoculate the dried surface of MHA plate by streaking the swab three times over the
entire agar surface; rotate the plate approximately 60 degrees each time to ensure an
even distribution of the inoculums (Figure 3).
4. Rim the plate with the swab to pick up any excess liquid (Figure 4).
5. Discard the swab into an appropriate container.
6. Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5
minutes, but no more than 15 minutes, for the surface of the agar plate to dry.
E. PLACEMENT OF THE ANTIBIOTIC DISKS

1. To add disks one at a time to the agar plate using forceps, place the MH plate on the
template (Fig. 7) provided in this procedure (Fig. 6a). Sterilize the forceps by cleaning
them with a sterile alcohol pad and allowing them to air dry or immersing the forceps in
alcohol then igniting.
2. Using the forceps carefully remove one disk from the cartridge (Fig. 6b).
3. Partially remove the lid of the petri dish. Place the disk on the plate over one of the dark
spots on the template and gently press the disk with the forceps to ensure complete
contact with the agar surface. Disks should not be placed closer than 24 mm (center to
center) on the MH agar plate. The template provided in this protocol (Fig. 7) maintains
the recommended 24 mm center to center spacing and allows the placement of up to 8
disks on the plate. Replace the lid to minimize exposure of the agar surface to room air
(Fig. 6c, d).
4. Continue to place one disk at a time onto the agar surface until all disks have been
placed
5. Once all disks are in place, replace the lid, invert the plates, and place them in a 35°C air
incubator for 16 to 18 hours.
F. MEASURING THE INHIBITION ZONE

1. Following incubation, measure the zone sizes to the nearest millimeter using a ruler or
caliper; include the diameter of the disk in the measurement (Fig. 8, 9b).
2. When measuring zone diameters, always round up to the next millimeter.
3. All measurements are made with the unaided eye while viewing the back of the petri
dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with
reflected light (Fig. 9a).
4. View the plate using a direct, vertical line of sight to avoid any parallax that may result in
misreading.
5. Record the zone size on the recording sheet.
6. If the placement of the disk or the size of the zone does not allow you to read the
diameter of the zone, measure from the center of the disk to a point on the
circumference of the zone where a distinct edge is present (the radius) and multiply the
measurement by 2 to determine the diameter (Fig. 10).
7. Growth up to the edge of the disk can be reported as a zone of 0 mm.
G. INTERPRETATION OF THE RESULTS

1. Using the published CLSI guidelines, determine the susceptibility or resistance of the
organism to each drug tested (Tables 1, 2, 3). Note that there are different charts for
different organisms. Abbreviated charts specific for the author’s suggested organisms
and antimicrobial disks to use are provided below.
2. 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.
3. The results of the Kirby-Bauer disk diffusion susceptibility test are reported only as
susceptible, intermediate, or resistant.
Figure 1. McFarland standards (left to right) 0.5, 1.0, 2.0, 3.0 positioned in front of a Wickerham
card. The bacterial suspension of the organism to be tested should be equivalent to the 0.5
McFarland standard when using Kirby-Bauer disk diffusion susceptibility test.
Figure 2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure
to remove the excess fluid. The swab should not be dripping wet.
Figure 3. Inoculate the dried surface of MHA plate by streaking the swab three times over the
entire agar surface; rotate the plate approximately 60 degrees each time to ensure an even
distribution of the inoculums
Figure 4. Rim the plate with the swab to pick up any excess liquid
REFERENCES:
Bauer, A. W., D. M. Perry, and W. M. M. Kirby. 1959. Single disc antibiotic sensitivity testing of
Staphylococci. A.M.A. Arch. Intern. Med. 104:208–216.
Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing
by a standardized single disk method. Am. J. Clin. Pathol. 36:493-496.
Clinical Laboratory Standards Institute. 2006. Performance standards for antimicrobial disk
susceptibility tests; Approved standard— 9th ed. CLSI document M2-A9. 26:1. Clinical
Laboratory Standards Institute, Wayne, PA.
Difco. 1984. Difco manual, 10th ed. Difco Laboratories, Detroit, MI.
Kirby, W. M. M., G. M. Yoshihara, K. S. Sundsted, and J. H. Warren. 1957. Clinical usefulness

of a single disc method for antibiotic sensitivity testing. Antibiotics Annu. 1956-1957:892.
Winn, Jr., W., et al. 2006. Konemann’s color atlas and diagnostic text of microbiology, 6th ed., p.
945–1021. Lippencott Williams & Wilkins Publishers, Philadelphia, PA.
Jorgensen, J. H., and J. D. Turnidge. 2007. Susceptibility test methods: dilution and disk
diffusion methods, p. 1152–1172. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry,
and M. A. Pfaller (ed.), Manual of clinical microbiology, 9th ed. ASM Press, Washington, D.C.

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What is the Kirby-Bauer disk diffusion susceptibility test?

What are the steps involved in performing the Kirby-Bauer disk diffusion susceptibility test?

INTRODUCTION:

Antimicrobial susceptibility testing is important in production of new drugs, epidemiology,

KIRBY –BAUER DISK DIFFUSION SUSCEPTIBILITY TEST

EXPECTED LEARNING OUTCOMES:

D. INOCULATION OF BACTERIAL SAMPLES INTO THE MH PLATE

E. PLACEMENT OF THE ANTIBIOTIC DISKS

F. MEASURING THE INHIBITION ZONE

G. INTERPRETATION OF THE RESULTS

Kirby, W. M. M., G. M. Yoshihara, K. S. Sundsted, and J. H. Warren. 1957. Clinical usefulness

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