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 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
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LABORATORY #12
Antibiotic Susceptibility Testing
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-tobatch 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
colonies of S. aureus from a plate of 18-24 hour growth on non-selected agar. The other
partner will select a well-isolated morphologically identical colonies of E. coli from a
plate of 18-24 hour growth on non-selected 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
will demonstrate this technique.
11. Close the plate and tap the bottom of the plate lightly against the edge of a tabletop to
ensure contact between the agar and the disks.
12. Place the plate in the 35°-37°C, non- CO2 incubator overnight.
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LABORATORY #12
Antibiotic Susceptibility Testing
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
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.
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LABORATORY #12
Antibiotic Susceptibility Testing
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
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.
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LABORATORY #12
Antibiotic Susceptibility Testing
Interpretation for Streptococcus Pneumoniae
*NCCLS interpretative standards for MICs(ug/mL)
S
Levofloxicin
Erythromycin
Vancomycin
Trim/Sulfa
Doripenem
<2
<0.25
<1
<32
<0.125
I
R
3-6
>8
0.5
>1
No interpretive criteria for “I” or “R”
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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LABORATORY #12
Antibiotic Susceptibility Testing
Kirby-Bauer Standard Antibiotic Sensitivity Zone Sizes
Staphylococci Zone Sizes
Resistant I
Sensitive
(S)
Resistant
I
NA
≥ 29 mm
≤ 13 mm
14-16
mm
≥ 17 mm
≤14 mm
15-22
mm
≥23mm
≤14 mm
15-22
mm
≥23mm
CPD10
≤ 17 mm
18-20
mm
≥ 21 mm
≤ 17 mm
18-20
mm
≥ 21 mm
Clarithromycin
CLR15
< 13mm
14-17
mm
> 18mm
NA
NA
NA
Ciproflozacin
CIP5
≤ 15 mm
16-20
mm
≥ 21 mm
≤ 15 mm
16-20
mm
≥ 21 mm
Erthromycin
E15
≤ 13 mm
14-22
mm
≥ 23 mm
NA
NA
NA
Nitrofurantoin
F/M300 ≤ 14 mm
15-16
mm
≥ 17 mm
≤ 14 mm
15-16
mm
≥ 17 mm
Ofloxacin
OFX5
≤12 mm
13-15
mm
≥16 mm
≤ 12 mm
13-15
mm
≥ 16 mm
Tetracycline
TE30
≤14 mm
15-18
mm
≥19 mm
≤ 14 mm
15-18
mm
≥ 19 mm
Trimethroprim/
SXT
≤10 mm
11-15
mm
≥16 mm
≤ 10 mm
11-15
mm
≥ 16 mm
Antibiotic
Symbol
Ampicillin
AM10
≤ 28 mm
Cefotaxime
CTX30
Cefpodoxime
Sulfamethoxazole
InterMediate
(I)
Enterobacteriaceae Zone Sizes
InterMediate
(I)
Sensitive
(S)
*Updated 7/2011
BD BBL Sensi-Disc Antimicrobial Susceptibility Test Discs Insert
MLAB 2434 – Laboratory 12 – Page 6
LABORATORY #12
Antibiotic Susceptibility Testing
Lab #12: Antibiotic Sensitivity
Kirby-Bauer Report Form
Points= 10
Name
______ Date
Antibiotic
Symbol
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/Sufamethoxazole
SXT
S. aureus Zone Size R, I or S E. coli Zone Size R, I, or S
(mm)
(mm)
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LABORATORY #12
Antibiotic Susceptibility Testing
Lab # 12:Etest Report Form
Points=5
Name
______ Date
Organism:______________________
Antibiotic
Vancomycin
Symbol
VA
Tolerance limits
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
Reading
R,I,S
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LABORATORY #12
Antibiotic Susceptibility Testing
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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LABORATORY #12
Antibiotic Susceptibility Testing
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.)
MLAB 2434 – Laboratory 12 – Page 10