LABORATORY #12

Antibiotic Susceptibility Testing

Laboratory #12
Antibiotic Susceptibility Testing
Skills= 15 Points

Objective:
At the completion of this laboratory, the student will be able to:
1. Correctly set up and read Kirby-Bauer antibiotic sensitivity tests on Staphylococcus aureus
and E. coli.
2. Correctly set up and interpret an Etest.
3. Discuss the principle behind the Etest.
4. Discuss the principle behind the MIC.

Materials: Two 100 mm Mueller-Hinton plates

Plate cultures of Staphylococcus aureus and E.coli
Sterile 0.85% saline
Sterile Swabs
McFarland 0.5 turbidity standard
Antibiotic sensitivity disks
Antibiotic disk dispenser
Incubator
Forceps

References:
1. Mahon and Manuselis, Textbook of Diagnostic Microbiology, Third Edition, Chapter 13
2. Clinical and Laboratory Standards Institutes (formerly NCCLS) Standards for Antimicrobial
Susceptibility (http://www.clsi.org)

Discussion:
The traditional laboratory test for in vitro antimicrobial susceptibility has been the antimicrobial
disk-agar diffusion procedure, the so-called disk method. Its simplicity, speed of performance,
economy, and reproducibility, (under standardized conditions) have made it useful.

Filter paper disks are impregnated with various antimicrobial agents of specific concentrations
and are carefully placed on an agar culture plate that has been inoculated with a standardized
culture of the bacterium to be tested. The broth culture is standardized by comparing the
turbidity of the culture to the turbidity of a 0.5 McFarland standard, a turbid solution made by
mixing barium chloride (BaCl2) and sulfuric acid (H2SO4). The antibiotic disks are usually kept
refrigerated to maintain viability. It is necessary to allow the disks to come to room

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temperature before placing them on the inoculated agar to avoid cold shocking the organism.
The disks should be placed on the inoculated agar within 15 minutes so that the organism has
not had a chance to begin growing and to avoid a false negative reaction.

The plate is incubated overnight and observed the following day for a zone of growth inhibition
around the disk containing the agent to which the organism is susceptible. Antibiotics causing
zones of inhibition require that the zone size be measured and interpreted in order to
determine the relative susceptibility or resistance of an organism. The size of the zone of
inhibition denotes the concentration point at which the antibiotic concentration fails to inhibit
the growth of the bacterium. The diameter of the zone of inhibition directly relates to the MIC.
The larger the diameter of the zone of inhibition for a given antibiotic and organism, the more
susceptible the organism. The resistant organism will have a smaller zone size. Antibiotic disks
with no zone of inhibition indicate that the antibiotic did not inhibit the growth of the organism
even at the highest concentration and the organism is resistant to the antibiotic.
The disk diffusion method has been standardized through the work of Kirby and Bauer.

Mueller-Hinton agar is chosen for routine susceptibility testing since it shows good batch-to-
batch uniformity and is low in tetracycline and sulfonamide inhibitors. With the addition of
animal blood, it will support the growth of most fastidious pathogens.

Kirby-Bauer Method

Procedure:
1. Working in pairs, each person will perform one Kirby-Bauer set-up.
2. Using a sterile swab, one person will select a well-isolated morphologically identical
colony of S. aureus from a plate of 18-24 hour growth on non-selective agar. The other
partner will select a well-isolated morphologically identical colony of E. coli from a plate
of 18-24 hour growth on non-selective agar.
3. Transfer growth to a tube of sterile 0.85% saline.
4. Emulsify and mix well.
5. Adjust turbidity using sterile saline to equal a 0.5 McFarland standard. Hold the two
tubes side by side up to a light source to compare turbidity.
6. Within 15 minutes of inoculum preparation, dip a sterile cotton-tipped swab into the
inoculums suspension and swirl to saturate the swab.
7. Press and rotate the swab several times against the side of the tube to remove excess
liquid.
8. Inoculate the Mueller-Hinton plate by spreading the organism suspension over the
entire surface with the swab. Rotate the plate approximately 90 degrees, and swab the
entire surface with the same swab. Rotate the plate another 90 degrees and swab the
surface a final time to ensure an even distribution of inoculum.
9. Allow the plate to dry at least 1 minute with the lid in place.
10. Open the lid, then apply the antibiotic sensitivity discs with the dispenser. The instructor

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will demonstrate this technique.

11. Tap the disks, once placed on the agar, to ensure contact between the agar and the
disks. This practice also prevents loosely seated disks from falling off the agar during
incubation.
12. Place the plate in the 35°-37°C, non- CO2 incubator overnight.
13. Using a metric ruler, measure the zones of inhibition (no growth zones) around each
antibiotic sensitivity disk from edge of growth through the disk to the opposite edge of
growth.
14. Record the zone sizes in millimeters (mm) on the Kirby-Bauer report form.
15. Then, indicate on the report form if the zone indicates the organism is “sensitive”,
“intermediate”, or “resistant” to each antibiotic, based on the Kirby-Bauer Standard
Antibiotic Sensitivity Zone chart at the end of this laboratory exercise.

E Test System
Principle:
The E-test is a quantitative MIC method for testing the antimicrobial susceptibility of fastidious
and non-fastidious organisms. Etest is used routinely for testing the MIC of Streptococcus
pneumonia and strep viridians group isolates recovered from blood cultures of endocarditis
patients and other streps as requested. One benefit of the Etest system is that the antimicrobial
menu is easily customized, however, it is expensive so it may not be extensively used.

The E-test strip is an inert plastic strip which has a numerical MIC scale printed on one side and
an antibiotic concentration gradient dried on the other side. The strips are applied to a
standardized inoculum of an organism swabbed onto the surface of an agar plate. The
antimicrobial agent diffuses into the agar, and a stable continuous antibiotic concentration
gradient is established along the sides of the strip. After incubation, an elliptical zone of
inhibition forms. The MIC is read where organism’s growth intersects the E-strip scale.
Materials:
Etest strips of designated test antimicrobials
Plate culture of S. pneumoniae
Sterile cotton-tipped swabs
McFarland 0.5 standard
Sterile pipettes
One 150 mm Mueller Hinton sheep blood agar (MHB plates)
0.85% saline
Forceps
Incubator, 35oC, CO2

Procedure:
1. Allow Etest strips to equilibrate to room temperature for 30 minutes prior to use.
2. Allow Mueller Hinton sheep blood agar plates to equilibrate to room temperature.
3. Using a sterile swab, select well-isolated morphologically identical colonies from a plate

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of 18-24 hour growth on non-selected agar.

4. Transfer growth to a tube of sterile 0.85% saline.
5. Emulsify and mix well.
6. Adjust turbidity using sterile saline to equal a 0.5 McFarland standard. Hold the two
tubes side by side up to a light source to compare turbidity.
a. NOTE: When testing mucoid Strep pneumonia or other mucoid or fastidious
organisms, the inoculum should be equivalent to McFarland #1 standard.
7. Within 15 minutes of inoculum preparation, dip a sterile cotton-tipped swab into the
inoculum suspension and swirl to saturate the swab.
8. Press and rotate the swab several times against the side of the tube to remove excess
liquid.
9. Inoculate the plate by spreading the organism suspension over the entire surface with
the swab. Rotate the plate approximately 90 degrees, and swab the entire surface with
the same swab. Rotate the plate another 90 degrees and swab the surface a final time
to ensure an even distribution of inoculum.
10. Allow the inoculum to dry on the plates for 10-15 minutes before proceeding. This step
is critical to proper performance.
11. Using forceps or fingers, grip the handle of the strip (E label end) and remove from the
package. DO NOT touch the antibiotic gradient.
12. Holding the strip at an angle to the plate, place the bottom edge of the strip against the
agar surface with the MIC scale facing upwards and the “E” label end pointing to the
outer edge of the plate.
13. Gradually allow the strip to come in contact with the agar, by anchoring the end with a
swab and slowly lowering the strip.
14. Make sure the strip is in complete contact with the agar surface. Remove air pockets
underneath by gently pressing with a swab, moving from the minimum concentration
upwards. Small bubbles under the strip will not affect the results. Once applied, the
position of the strip cannot be changed due to the immediate release of the antibiotic
from the underside of the strip on the agar.
15. When using a 150-mm plate, place up to six Etest strips on the plate. Place the strips an
equal distance apart, radiating from the center of the plate like spokes of a wheel with
the “E” end of the strip pointing outwards.
16. If the strip is accidentally placed upside down, carefully pick it up by the “E” end, turn it
over, and position it properly on the agar surface.
17. If the strip touches the surface of the bench or another object, it can still be used as long
as it does not come in contact with moisture.
18. Invert the plates and incubate for 18-24 hours in a 35OC CO2 incubator within 1 hour of
strip application.

Interpretation/Reading plates:
1. Read plates only if the lawn of growth is confluent or nearly confluent.
2. Remove the lid and use reflected light to read the MIC at the point where the growth

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intersects the strip.

3. If the growth does not intersect the strip, record the MIC as less than the lowest
concentration of antimicrobial agent on the Etest strip.
4. If growth occurs along the entire strip (no ellipse seen), record the MIC as greater than
the highest value on the scale.
5. If growth intersects the strip, record the MIC as the numerical value at the point of
intersection. For MICs that fall between markings, record the result as the higher value.
6. Enter the numerical MIC value in the report form for the designated Etest strip.
7. Using the MIC value, and the chart below, interpret as “S, I, or R” based on organism
specific database definitions.

Interpretation for Streptococcus Pneumoniae

*NCCLS interpretative standards for MICs(ug/mL)

S I R
Levofloxicin <2 3-6 >8
Erythromycin <0.25 0.5 >1
Vancomycin <1 No interpretive criteria for “I” or “R”
Trim/Sulfa <32
Doripenem <0.125

MIC (Minimal Inhibitory Concentration)

The MIC is the lowest concentration of a drug in mg/ml that inhibits the growth of a
microorganism.

Read pages 281-284 in the textbook related to “Dilution Methods” for sensitivity testing and
Minimum Inhibitory Concentration and answer the questions at the end of this laboratory
exercise. Turn the answers in to complete points for this laboratory.

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Kirby-Bauer Standard Antibiotic Sensitivity Zone Sizes

Staphylococci Zone Sizes Enterobacteriaceae Zone Sizes

Antibiotic Symbol Resistant I Inter- Sensitive Resistant Inter- Sensitive
Mediate (S) I Mediate (S)
(I) (I)

Ampicillin AM10 ≤ 28 mm NA ≥ 29 mm ≤ 13 mm 14-16 ≥ 17 mm

mm

Cefotaxime CTX30 ≤14 mm 15-22 ≥23mm ≤14 mm 15-22 ≥23mm

mm mm

Cefpodoxime CPD10 ≤ 17 mm 18-20 ≥ 21 mm ≤ 17 mm 18-20 ≥ 21 mm

mm mm

Clarithromycin CLR15 < 13mm 14-17 > 18mm NA NA NA

mm

Ciproflozacin CIP5 ≤ 15 mm 16-20 ≥ 21 mm ≤ 15 mm 16-20 ≥ 21 mm

mm mm

Erthromycin E15 ≤ 13 mm 14-22 ≥ 23 mm NA NA NA

mm

Nitrofurantoin F/M300 ≤ 14 mm 15-16 ≥ 17 mm ≤ 14 mm 15-16 ≥ 17 mm

mm mm

Ofloxacin OFX5 ≤12 mm 13-15 ≥16 mm ≤ 12 mm 13-15 ≥ 16 mm

mm mm

Tetracycline TE30 ≤14 mm 15-18 ≥19 mm ≤ 14 mm 15-18 ≥ 19 mm

mm mm

Trimethroprim/ SXT ≤10 mm 11-15 ≥16 mm ≤ 10 mm 11-15 ≥ 16 mm

Sulfamethoxazole mm mm

*Updated 7/2011

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BD BBL Sensi-Disc Antimicrobial Susceptibility Test Discs Insert

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Lab #12: Antibiotic Sensitivity

Kirby-Bauer Report Form
Points= 10

Name ______ Date

Antibiotic Symbol S. aureus Zone Size R, I or S E. coli Zone Size R, I, or S

(mm) (mm)

Ampicillin AM10

Cefotaxime CTX30

Cefoxitin FOX30

Cefpodoxime CPD10

Clarithromycin CLR15

Ciproflozacin CIP5

Erythromycin E15

Nitrofurantoin F/M300

Norfloxacin NOR-10

Ofloxacin OFX5

Tetracycline TE30

Trimmethoprim/Sufamethoxazol SXT
e

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Lab # 12:Etest Report Form

Points=5

Name ______ Date

Organism:______________________

Antibiotic Symbol Tolerance limits Reading R,I,S

Vancomycin VA 0.12-0.5

Levofloxacin LE 0.5-2

Erythromycin EM 0.03-0.25

Trim/Sulfa TS 0.002-32

Doripenem DOR 0.032-0.125

Ceftriaxone TX 0.03-0.12

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Laboratory #12: Antibiotic Susceptibility Testing

Study Questions
Points= 13

Name Date

1. Describe an E test plastic strip. How are they placed on an agar plate? How do they compare
in price to standard antibiotic sensitivity disks? For which types of organisms are the E test
especially useful? (2 pts)

2. What is MIC? Briefly explain how an MIC is performed. (2 pts)

3. What is CLSI (http://www.clsi.org) and what part does it play in antibiotic sensitivity testing?
(2 pts)

4. When performing microdilution MIC testing, what two control wells are added on each tray
and why?(3 pts)

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5. What is a breakpoint panel? (1 pt)

6. Briefly describe “trailing” and “skipped wells” when performing MICs. How should each be
handled? (3 pts.)

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