Kirby Bauer Method

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EXPERIMENT 9

Kirby-Bauer Antimicrobial Susceptibility Test Abenoja, Asto, Baquiran, Briones

ABSTRACT

Kirby-Bauer Antimicrobial test is used in determining a species’ susceptibility to aspecific agent, mostly antibiotics. The goal of this study was to conduct the test toidentify the susceptibility by using disk diffusion method to apply the susceptibility ofselected cultures using Ampicillin, Vancomycin, Kanamycin, Streptomycin, andHydrogen Peroxide, and to distinguish the range of their activity. An antibiotic disk andfilter paper soaked in hydrogen peroxide was suspended in Mueller-Hinton agar plateswhich were then incubated. The zones of inhibition were measured and it was foundthat the first three unknown having a gram positive ID were resistant to ampicillin butsusceptible to vancomycin, white they were resistant to intermediate reactions tokanamycin, and susceptible to intermediate reaction to streptomycin. The fourthunknown has a gram negative ID and did not exhibit growth.

INTRODUCTION

Microorganisms are ubiquitous. Their high reproduction and mutation rate gave them

the ability to thrive everywhere. For pathogens, this resulted in mechanisms that give

them a higher chance of infecting a host. Due to this, various bactericidal/static agents

were developed. The everyday use of bacteriostatic and bactericidal agents to treat

infections or to disinfect surfaces gave rise to the need to test this agent’s effectiveness

in the laboratory. Kirby-Bauer Antimicrobial Disk Susceptibility Test is one of the most-

utilized methods in determining a species’ susceptibility to a specific agent, especially

antibiotic.

This method utilizes the use of antibiotic disk or a filter paper soaked in a

bactericidal/static agent suspended on the Mueller Hinton Agar. Organism is added

upon transfer of media, by combining hard agar and soft agar with the organism on it.

The disks were later added after the media solidifies. Mueller Hinton Agar is considered

as best media to use for the routine susceptibility testing since it is produces batch-to-



batch reproducibility, low concentration of inhibitors of sulphonamide, trimethoprim and

tetracyclines and produce satisfactory results for most of the non-fastidious pathogens.

Also, there has been a considerably large body of data and experience recorded by

microbiologists concerning its satisfactory results in susceptibility tests performed with

this medium[1]. Organism’s susceptibility is determined when a zone of inhibition is

visible.

In this experiment, Kirby-Bauer Antimicrobial Disk Susceptibility Test was

conducted on four selected cultures previously isolated, using four different kinds of

antibiotics and bactericidal/static agents and one germicidal agent, namely hydrogen

peroxide to demonstrate the inhibitory effect of some chemical substances to bacterial

cells, to distinguish the range of activity of some antibiotics and to apply the disk

diffusion method in evaluating the susceptibility of bacteria to different antimicrobial

chemicals.

METHODOLOGY

There were three main steps for this study: first, the preparation of culture media,

second was the inoculation of test plates and third was the application of disks to

inoculated agar plates. The culture media necessary for this experiment is the Mueller-

Hinton (MH) base agar and soft agar. The two types of media were simultaneously

prepared: base agar plate was made by dissolving approximately 11.5 g of Mueller-

Hinton agar in 300 mL distilled water (3.8/100mL) in an Erlenmeyer flask, and the soft

agar was prepared following the same procedure, but only with half the weight of the

original mixture (0.94 g/ 100 mL). These mixtures were then subjected to autoclaving



and then subsequently poured the prepared base agar to 8 disposable petri plates, with

only ⅓ of the volume of each plate being filled. This was set aside to solidify. Next was

the inoculation of test plates in which the broth culture of the chosen bacteria, prepared

prior to the experiment, was mixed with soft agar. The mixture was then poured to the

plate containing the solidified MH base agar. Finally, the application of disks to

inoculated agar plates. This was done by placing antibiotic disks of Kanamycin,

Vancomycin, Ampicillin, and Streptomycin and plates soaked in hydrogen peroxide (the

germicidal agent chosen) and water adjacently on the surface of the medium, with

plates soaked in water as the negative control, noting that the distances were ample

enough for the formation of the Zones of Inhibition. The plates were then incubated for

24 hours at 35 degrees Celsius and the zones of inhibition were then noted by

subtracting the diameter of the disk from the total diameter of visible zone of inhibition

and subsequently compared with the determined and established diameter size of zone

of inhibition for susceptibility.

RESULTS AND DISCUSSIONS

In this study, the antimicrobial activity of four selected antibiotics namely,

Ampicillin (Amp), Vancomycin (Van), Kanamycin (K) and Streptomycin (S), and one

disinfecting agent, hydrogen peroxide, were evaluated using four unknown microbial

species, with samples 1-3 being gram positive and 4 being gram negative, using the

sensitivity disk Kirby-Bauer method. Kirby- Bauer test is a standardized method used to

determine if a particular strain of bacterial species is susceptible or resistant to



antibiotics based on the size of the zone of inhibition respective for the antibiotics and

bacterial sample used which is evident in a form of a clearing in a lawn of bacteria.

Here, Mueller-Hinton agar is used in antibiotic susceptibility test as the base agar

media. The starch in the Mueller- Hinton agar mediates the diffusion of the antibiotic,

has low concentration in sulfonamide, trimethoprim, and tetracycline inhibitors, thus

formation of thymine- dimers will be inhibited [2]. After incubation on Mueller-Hinton agar

plate, the total diameter of the visible zone of inhibition is measured in millimeters which

is shown in the table below.

Table 1. Measured diameters (in millimeter) of the total visible zones of inhibitionof each unknown sample

Unknown 1 Unknown 2 Unknown 3 Unknown 4

Diskdiameter (mm)

Trial 1 (mm)

Trial 2 (mm)

Trial 1(mm)

Trial 2 (mm)

Trial 1(mm)

Trial 2(mm)

Trial 1(mm)

Trial 2 (mm)

Amp 6 0 0 0 6 6 0 no growth

Va 6 21 20 22 20 21 23

K 6 20 19 23 20 22 22

S 6 19 20 23 22 22 20

H2O2 7 20 16 24 27 20 20

H2O 7 0 0 0 0 0 0

As observed above, there was absolutely no bacterial lawn clearing to which the

disk soaked in water was placed, indicating then that water alone cannot prevent

disinfection. Also, it is important to mention that due to improper handling of sample, the

unknown specimen 4 had not been cultured on the media, thus no growth was observed

at all.



The measured disk diameters of each disk of antibiotics, antimicrobial agent and

water were then subtracted to the measured total visible zones of inhibition to give the

precise zones of inhibition which are presented in the Table 2.

Table 2. Zones of inhibition measured for each unknown

Unknown 1 Unknown 2 Unknown 3

Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2

Amp - - - - - -

Va 15mm 14 mm 16 mm 14 mm 15 mm 17 mm

K 14 mm 13 mm 17 mm 14 mm 16 mm 16 mm

S 13 mm 14 mm 17 mm 16 mm 16 mm 14 mm

H2O2 13 mm 9 mm 14 mm 20 mm 14 mm 14 mm

H2O - - - - - -

The table above shows the measurement of the zones of inhibition exhibited by

the unknown bacterial species. Notably, they exhibited different size which may be due

to the bacteria’s susceptibility, the diffusion rate of the antibiotic, the depth of the agar,

pH, size of the inoculated organism, and the presence of other metals [3]. Organisms

susceptible to the antibiotic are unable to grow near the disc, whereas there will be an

observed presence of growth adjacent to the disc if they are resistant. The molecular

weight, solubility properties of the antibiotic, and concentration affects the diffusion rate

of the antibiotic which results to a unique breakpoint zone size indicating its

susceptibility [3].As the disk is placed on the medium, water is first absorbed into the disk

from the agar, then the antimicrobial compound follows but its rate of diffusion is not as



fast as the rate of its extraction out of the disk, thus its concentration is highest when

closest to the lead and is reduced as the distance increases. Also, the higher its

molecular weight, the slower its rate of diffusion. In order to effectively test the

susceptibility of a bacteria in a dehydrated media, the plates must be poured to a depth

of 4mm. Shallow plates will yield to false susceptibility as it results to a wider range of

diffusion of the antimicrobial compound than it should, creating a larger zone of

inhibition. Furthermore, the pH should be maintained at 7.2 and 7.4 at room

temperature to ensure the quality of the drugs, while the number of inoculated

organisms should be standardized to avoid errors since smaller number of inoculum

would lead to a false susceptibility of the microorganism. Lastly, the presence of other

metals like excess zinc will affect the bacteria and the quality of the drug on the agar

plate.

To assess the susceptibility, a table 3[9] is provided below to standardize

interpretation of the results.



Table 3. Interpretation of zones of inhibition (in mm) for Kirby-Bauer antibioticsusceptibility test

The zone of inhibition also directly relates to the bacteria’s susceptibility. A bigger zone

of inhibition means that the bacteria is susceptible at the concentration tested while a

smaller zone of inhibition suggests that the antibiotic is not effective. The intermediate

result suggests that the substance is effective in higher concentrations [4]. Using the

table given above, the following has been deduced:



Table 4. Reaction of the unknown bacterial samples to some antibiotics,hydrogen peroxide and water

Unknown 1 Unknown 2 Unknown3

Amp Resistant Resistant Resistant

Va Susceptible Susceptible Susceptible

K Resistant toIntermediate

Intermediate Intermediate

S Intermediate Susceptible Intermediate tosusceptible

H2O2 Susceptible Susceptible Susceptible

H2O Resistant Resistant Resistant

Antimicrobial drugs act either by damaging an intracellular structure or inhibiting

a vital metabolic function but these drugs mainly have five actions: inhibition of cell wall

synthesis, disruption of cell membrane, inhibition of protein synthesis, inhibition of

nucleic acid synthesis and the inhibition of folic acid synthesis [5]

Ampicillin (Amp) diffuses into most body tissues and fluids readily. They target

the cell wall of bacteria by blocking peptidoglycan synthesis at the final step of cross-

linking mesh, thereby unable to maintain their cell wall [5]. Likewise, Vancomycin(Va)

inhibits the synthesis of cell wall by inhibiting the peptidoglycan synthesis. It is a drug

from the class of glycopeptide antibiotics. They are essential for the control of most

gram positive bacteria. Kanamycin(K) and streptomycin(S) inhibit protein synthesis of

bacteria. They came from the class of aminoglycoside bacteriocidal antibiotics.

Aminoglycosides were discovered in deliberate, intensive search for antimicrobials that

are effective against gram positive bacteria [5]. They are composed of amino sugar

molecules connected to one another by glycosidic bonds and have a selective toxicity.



They bind irreversibly to the 30S subunit of ribosome which in turn will slow protein

synthesis thereby producing a faulty protein [5]. Toxicity is increased due to the fact that

aminoglycoside molecules are actively transported in the cells, but they penetrate

human cells with great difficulty.

As seen above, the unknown samples were resistant to ampicillin, having a disk

concentration of 10μg and a molecular weight of 349.406 g/mol, while they exhibited

susceptibility to vancomycin, with a disk concentration of 30μg and a molecular weight

of 1485.71454 g/mol. It is expected that with the same targets (cell wall synthesis) of

both antibiotic, they are to have same reactions from the gram positive species yet differ

only in the diameter of zones of inhibition as Vancomycin has higher disk concentration

than Ampicillin (refer to Table 3) . Yet they were all resistant to Amp and susceptible to

Va. This can be attributed to two factors: one, is the use of expired disks that made

them resistant to Amp adn two, is the higher disk concentration of Va. The unknown

samples, however, exhibited different responses to Kanamycin and Streptomycin.

These differences in reaction to Kanamycin and Streptomycin may also be due to their

differences in molecular weights, with S having 581.574 g/mol while K having 484. 499

g/mol, and in disk concentration with S having a lower one than K. Thus, it is expected

that K would be more effective than S in terms of zone of inhibition, and both be more

toxic to bacteria than Amp and Va with their mode of action or target. This erroneous

result may also be affected by the expiration dates of such antibiotics.

As for the reaction to hydrogen peroxide, it must be understood that during the

detoxification of superoxide radicals by superoxide dismutases, hydrogen peroxide is

formed. This contains peroxide anion, making it an antimicrobial agent. However, there



are enzymes which can in turn, detoxify it -- catalase and peroxidase, both in aerobic

bacteria. Meanwhile, obligate anaerobes either lack or have insufficient amount of these

enzymes, thus becoming susceptible to them[7]. Looking at Table 2, a 9-14 millimeter

diameter formed around the disks soaked with hydrogen peroxide among the unknown

species, thus categorizing them to be susceptible in Table 4 and further indicating that

they are obligate anaerobic bacterial species.

The limitation of this test, however, is that it cannot differentiate whether the the

zone of inhibition was either due to death or inhibition. In order to determine if it was in

either state, a sample from the zone of inhibition should be inoculated into a nutrient

broth, and incubated. If there is growth, then the action of the antibiotic was probably to

inhibit than to cause death.

CONCLUSION

The study was done using four previously isolated and selected cultures, the first three

having gram positive ID and the fourth being gram negative, were tested using Ampicillin,

Vancomycin, Kanamycin, Streptomycin, water, and hydrogen peroxide to demonstrate the

inhibitory effect of chemical substances to bacterial cells. The range of activity of the antibiotics,

and the antimicrobial agent using disk diffusion method in evaluating their susceptibility were

distinguished. From the three unknown samples examined, it was found that they were resistant

to ampicillin but susceptible to vancomycin while resistant to intermediate reactions to

Kanamycin and susceptible to intermediate reaction to Streptomycin. It is recommended that

viable antibiotic disks rather than expired ones be used to prevent erroneous results. It is further

recommended for future studies that determining whether the zone of inhibition is bacteriostatic

or bacteriocidal be done as a confirmatory test, and other antibiotics will be used. Moreover, the



samples to be used should be a known pathogenic microorganism to determine the antibiotics’

effectivity.

REFERENCES

[1]vlab.amrita.edu,. (2013). Antibiotic Susceptibility Testing. Retrieved 25 November

2015, from vlab.amrita.edu/?sub=3&brch=73&sim=1628&cnt=1

[2]Retrieved November 26, 2015 from

https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MuellerHintonMed.ht

m

[3] Factors that Affect the Zone of Inhibition and Zone of Inhibition Analysis.2011. Retrieved on

November 25, 2015 from http://hubpages.com/education/Zone-of-Inhibition-Testing

[4] Matros,L and Wheeler,T . 2001. Microbiology Guide to Interpreting MIC (Minimum

Inhibitory Concentration).IVS Sacramento.

[5] Ingraham. (1995). Introduction to Microbiology. Wadsworth Publishing Company.

[6] Linley, E. et al. (2012). Use of Hydrogen Peroxide as Biocide: New Considerations of its

Biocidal Action. Journal of Advanced Microbial Chemotherapy. Retrieved November 26,

2015 from http://jac.oxfordjournals.org/content/early/2012/04/23/jac.dks129.full.pdf

[7]. Bauman, Robert.2014. Microbiology with diseases by Taxonomy, 4th ed .New York: Pearson

[8] Hudzicki, Jan. 2009. Kirby-Bauer Disk Diffusion Susceptibility Test Protocol . American Society

of Microbiology. Retrieved from: www.microbelibrary.com

[9] Kirby-Bauer Antimicrobial Disk Susceptibility Test Protocol

APPENDIX

https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/MuellerHintonMed.htm



http://hubpages.com/education/Zone-of-Inhibition-Testing



http://jac.oxfordjournals.org/content/early/2012/04/23/jac.dks129.full.pdf



http://www.microbelibrary.com/










Fig.1 Showing the zones of inhibition by selected antibiotics and H2O2 on unknown 1.

Fig.2 Showing two sample trials of unknown 2.



Fig. 3 Showing the susceptibility of unknown 3, sample 1 and 2 to selected antibiotics

Fig. 4 Showing the reaction of unknown 4

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