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The Evaluation of Antibiotics Using Kirby

Bauer Disk Diffusion Method

ACKNOWLEDGMENTS: Source document adapted from: A Laboratory Manual for Microbioloy! T"irded#! by $o"n M# Lar%in! &it" 'ome addition' by (ut" A# Gyure for W)ST( &or%'"op $uly *++,! We'ternCT State -ni.er'ity-(L 'ource:"ttp://&&&a%'manfoundation#or/lab'/l'u/antibio#"tmlNote: T"e lab i' part of t"e Wa%'man 0oundation collection of microbioloy re'ource' for K12* teac"er'!an e3cellent re'ource! 'tartin pae found at:"ttp://&&&a%'manfoundation#or/"tml/re'ource4%2*#"tml5"oto of plate abo.e courte'y of -ni.er'ity of Ne& Sout" W"ale' Medical Sc"ool &eb'ite#

INTRODUCTION: Antibiotics are chemicals that are produced by living organisms which, even in minuteamounts, inhibit the growth of or kill another organism. While thousands of them have been discoveredsince Sir Alexander Fleming observed the inhibitory activity of Penicillium on Staphylococcus in !"!,

most are not useful medically because of undesirable toxicity or side effects. A simple method usingpaper discs saturated with the chemicals to be tested can be used to determine the resistance orsensitivity of an organism to different antibiotics. Following inoculation of an agar medium, discs areplaced onto the surface of the medium so that the antibiotic will diffuse into the medium. #nhibition of theorganism is evident following incubation as a clear region around the disc, called a $%one of inhibition$, inwhich no growth has occurred.

&he si%e of the %one of inhibition is determined by the type of medium used, the solubility and rate ofdiffusion of the antibiotic, the amount of inoculum, as well as the effect of the antibiotic. &his experimentsimulates a commonly used clinical techni'ue called the (irby)*auer method in which exact specificationsmust be followed. +umerous antibiotics are tested for usefulness against the actual organism causing apatients illness. -edical laboratory personnel select the antibiotic discs tested based upon the site andtype of infection. Following incubation and measurement of the %one si%es, a chart is consulted which

indicates whether the diameter of the %one of inhibition for an antibiotic indicates that it would be effectivefor use in treating the patients infection.

Materials per group of 4 students:

plates of &rypticase Soy Agar &SA/ or +utrient Agar +A/

sterile swabs

0vernight broth cultures of Stap"ylococcu' aureu'Sa/, and 5'eudomona' aeruino'aPa/ or

*S1 level organisms such as Micrococcu' luteu'! E'c"eric"ia coli! Stap"ylococcu' epidermidi',

http://www.waksmanfoundation.org/labs/lsu/antibio.htmlhttp://www.waksmanfoundation.org/labs/lsu/antibio.htmlhttp://www.waksmanfoundation.org/html/resource_k12.htmlhttp://www.waksmanfoundation.org/labs/lsu/antibio.htmlhttp://www.waksmanfoundation.org/html/resource_k12.html

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Materials to be shared by the class:

2ispensers of antibiotic discs

*eakers of disinfectant

Sterile toothpicks or applicator sticks

Protocol:

. 2ip one swab into the broth of 5'eudomona'.". Swab the entire surface of one plate labeled, $Pa$. 3o over the plate at least twice in each

direction. 2iscard the swab in the beaker of disinfectant provided.4. 5epeat this procedure for the Stap"ylococcu'.6. Add the antibiotic discs to each plate using the dispenser. 5emove the cover of the petri dish and

set the dispenser over the plate. #f the dispenser is not dispensing discs, consult the instructor.7. 3ently tap each antibiotic disc onto the surface of the agar with a sterile stick or toothpick to

assure good contact. 2iscard toothpicks in the disinfectant.8. #ncubate plates at 49:; until the next lab period.9. :xamine each plate for the presence of %ones of inhibition. mm ")6 mm 7 mmAmpicillin A- mm ")6 mm 7 mm3entamicin 3- " mm 4)6 mm 7 mm:rythromycin : 4 mm 6)"" mm "4 mm;hloramphenicol ; " mm 4)9 mm < mm&etracycline &e 6 mm 7)< mm ! mm

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Teacher"s instructions for The Evaluation of

Antibiotics&he teacher must inoculate tubes of &rypticase Soy broth or nutrient broth/ with Stap"ylococcu' aureu'and 5'eudomona' aeruino'aor other culture "6)6< hours before the class. 5emember to practice yourbest aseptic techni'ue while inoculating the cultures. &he cultures will be incubated at about 49?; until

needed.While the cultures can be used for more than one class, the potential for contamination of the culture willincrease with use. For this reason, you are provided four tubes of broth per class. Per class, inoculate twotubes with Stap"ylococcu' aureu'and two tubes with 5'eudomona' aeruino'aor other organism used.

Procedure for #a$ing the starting cultures:

. Start with sterile tubes of &rypticase Soy broth or +utrient *roth. #t is safest to A@&0;1A:

media but media boiled for > minutes or so will work for the procedure. *e sure to transfer mediacarefully and sterilely into the tubes, which would be best if disposable plastic purchased sterile/.1abel the tubes with names of organisms. Again, we suggest inoculating a separate starting tubefor each group of students and for each class.

". 0rgani%e your stock plates of organisms stored at 5& or in refrigerator with parafilm seals or in asealed box with humidity source/.

4. ;hoose one of the plates. @se a sterile disposable inoculation loop to place a visible amount ofthe bacteria from the surface of the plate into the correctly labeled tube of broth. Do not set theloop do%n. Bou will use the same loop to inoculate other tubes of broth with the same bacterium.2iscard of the loop in the bioha%ard bag or your beaker of bleach or disinfectant. ;ultures can beinoculated with sterile sticks but a disposable loop is simpler.

6. Snap or screw all the tube caps down tight. Colding the tube Dust below the cap, thump thebottom of the tube with your other hand to suspend the bacteria in the broth. 1oosen caps slightly.

7. #ncubate the tubes upright in the rack at approximately 49?; until class. @sually "6)6< hours isade'uate for abundant growth. &he broth in the tubes should be cloudy if the bacteria grew.

Some excellent supplementary PC0&0S of results of disk diffusion assay, from Albert :instein ;ollege of-edicineE httpEgold.aecom.yu.eduidmicrokirbybauer.htm

&NRIC'M&NT:

. An added activity from AS- -icrobelibrary is provided, allowing students to use statistical methods tocompare replicate samples from multiple groupsE@sing a 2isk 2iffusion Assay to #ntroduce Statistical -ethods

". Also see ;ultured #n'uiry, an article from +S&A Dournal. &his describes how to develop a true in'uirybased laboratory allowing students to test their own compounds for antibiotic effects on microbes.

http://gold.aecom.yu.edu/id/micro/kirbybauer.htmhttp://gold.aecom.yu.edu/id/micro/kirbybauer.htm