BIOL 3702L - Kirby-Bauer Method Fall 2020

Dr. Cooper 1

BIOL 3702L:Kirby-Bauer

Susceptibility Testing

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Purpose

• Understand the principle of the antibiotic susceptibility test;• Properly conduct the antibiotic

susceptibility test;• Determine the validity of the test using

known standards;• Correctly operate instrumentation; and• Accurately interpret the results of this

exercise.Copyright © C. R. Cooper, Jr.

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Today’s Lab Work

• Each group will perform one assay using either a different bacterial species.• Escherichia coli ATCC 25922 and

Staphylococcus aureus ATCC 25923 will serve as standard.• CAUTION: HANDLE ALL BACTERIAL STRAINS

APPROPRIATELY! Some are known pathogens, but are harmless if handled properly.

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2017/microbiologist-holding-a-petri-dish-with-bacteria-2/

3Copyright © C. R. Cooper, Jr.

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Kirby Bauer Method

https://youtu.be/Il9vcYZlVs8

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Today’s Lab Work (cont.)

• For each microbe, swab a MHA plate in three directions being sure to cover the edge of the plate.• Use dispenser to place antibiotic discs on

surface of MHA plate. BE SURE DISPENSER BUTTON IS IN LOCKED POSITION!!! If not, it can be easily broken!• Incubate 16-18 hours (ideally) at 37°C, but

return no later than 24 hours to make readings.

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2017/microbiologist-holding-a-petri-dish-with-bacteria-2/

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Today’s Lab Work (cont.)

• Record the zone diameter for each antibiotic disc tested for each strain.

• In this case, the diameter is 26 mm.Copyright © C. R. Cooper, Jr.

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BIOL 3702L - Kirby-Bauer Method Fall 2020

Dr. Cooper 2

Today’s Lab Work (cont.)

• Use the chart provided to make a determination as to susceptibility or resistance for this particular drug.• Record your data and your interpretation,

being sure that the standard strains (E. coli ATCC 25922 and S. aureus ATCC 25923) fall within the previously prescribed range.

Copyright © C. R. Cooper, Jr.https://fems-microbiology.org/international-womens-day-8-march-

2017/microbiologist-holding-a-petri-dish-with-bacteria-2/

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Today’s Lab Work (cont.)• Using the prior reading of 26 mm, presume

that the standards fell in the appropriate range, and assuming the drug used is ampicillin, what is your interpretation?

Copyright © C. R. Cooper, Jr.https://fems-microbiology.org/international-womens-day-8-march-

2017/microbiologist-holding-a-petri-dish-with-bacteria-2/

Antim icrobial AgentZone Diam eter Interpretative

Standards (m m )

Standard Control Strain

Response (m m )

E. coli

(ATCC 25922)

S. aureus

(ATCC 25923)

Am picillin Resistant Intermediate Susceptible 16-22 27-35

For Enterobacteriaceae ≤ 13 14-16 ≥ 17

For Staphylococcus species ≤ 28 ≥ 29

For Enterococcus species ≤ 16 ≥ 17

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Lab Report Expectations

• Complete each of the report sheets for this exercise. • Share your data with the rest of the class. The

data will be collected at the next laboratory session.• Answers to discussion questions must be clear,

concise, and grammatically sound.• Retain these laboratory exercise reports. Your

laboratory instructor may not call for them at this time, but may do so in the near future.

Copyright © C. R. Cooper, Jr.

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Questions?

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