DATE- 29.03.

2023

EXPERIMENT 8 – ANTIBIOTIC SENSITIVITY TEST

AIM: To detect the sensitivity of microorganisms to different antibiotics by Kirby-Bauer

Antibiotic Sensitivity Test.

PRINCIPLE: The available chemotherapeutic agents vary in their scope of antimicrobial activity.
Some have a limited spectrum of activity, being effective against only one group of
microorganisms. Others exhibit broad-spectrum activity against a wide range of microorganisms.
The drug susceptibilities of many pathogenic microorganisms are known, but it is sometimes
necessary to test several agents to determine the drug of choice.
A standardized filter paper disc-agar diffusion procedure, known as Kirby-Bauer method, is
frequently used to determine the drug susceptibility of microorganisms isolated from infectious
processes.
➢ Ampicillin, commonly known as a broad-spectrum penicillin, is a type of amino penicillin,
a semisynthetic group of β-lactams that are developed for effectiveness against both gram-
negative and gram-positive organisms.
➢ Tetracycline classify as protein synthesis inhibitor antibiotics and are broad-spectrum
agents, exhibiting activity against a wide range of gram-positive and gram-negative
bacteria, atypical organisms such as mycoplasmas and protozoan parasites.
➢ Streptomycin is an aminoglycoside and has a broad spectrum of antibacterial activity. It
is effective against most Gram-negative and a few Gram-positive bacteria; staphylococci,
streptococci and pneumococci.

MATERIALS REQUIRED:
(a) Liquid Cultures of Escherichia coli and Pseudomonas sp.
NAME CONCENTRATION
Ampicillin 10 µg/disc
Tetracycline 30 µg/disc
Streptomycin 10 µg/disc
(a) Mueller-Hinton Agar medium plates.
(a) Antimicrobial discs:
(b) EQUIPMENT: Laminar Air Flow cabinet, BOD-Incubator shaker
(c) OTHER REQUIREMENTS: Forceps, Spirit lamp, sterile cotton, sterile micropipette
tips, glass spreader, glassware marker, alcohol, standard millimetre ruler.

PROCEDURE:
(a) The petri plates with Mueller-Hinton Agar medium were labelled with the names of the
respective microorganisms using a glassware marker within the Laminar Air Flow cabinet
hood.
(b) The Mueller-Hinton Agar medium plates were opened.
(c) The culture tubes containing the cultures of microorganisms were opened and the mouths
of the tubes were flamed using a spirit lamp.
 (d) Using the pipetting device and sterile micropipette tips, 100 µl of each bacterial culture
was dispensed at the centre of the respective medium plates.
(e) A glass spreader was taken, dipped in alcohol and flamed (made sterile), and then allowed
to cool. It was applied on the medium plate to spread the culture, so that the bacterial
growth is uniform. The same process was performed for the other microorganism culture.
(f) A forceps was taken and flamed, and then allowed to cool. Using it the antimicrobial discs
were distributed in equal distances on the culture plates of both the microorganisms.
(g) Each disc was then gently pressed by the sterile forceps so that they adhere to the surface
of the medium.
(h) Both the culture plates were then placed in an upright position and incubated at 37°C for
24 to 48 hours.

OBSERVATIONS: After incubation at 37°C for 24 to 48 hours was complete, the culture plates
were taken out and the diameters of the zones of inhibition formed around the antimicrobial discs
were measured for both the microorganisms and a comparative analysis on antimicrobial
sensitivity by both the microorganisms were done. The diameters of the zones of inhibition as
measured are tabulated as follows:

ANTIBIOTIC E. coli Pseudomonas

Average Zone size (in mm) Average Zone size (in mm)
Ampicillin 9.4 0
Streptomycin 19.4 22.4
Tetracycline 17.4 8.4

CONCLUSION: Lesser is the diameter of zone of inhibition, more is the resistance of that
microorganism for that antibiotic or chemotherapeutic agent. From the table above, the
following conclusions can be made.
➢ Pseudomonas is more resistant to Ampicillin and Tetracycline than E. coli and more
sensitive to Streptomycin than E. coli.
➢ Escherichia coli is more sensitive to Tetracycline and Ampicillin than Pseudomonas and
more resistant to Streptomycin than Pseudomonas.
ANTIBIOTIC SENSITIVITY TEST ON Pseudomonas sp.

ANTIBIOTIC SENSITIVITY TEST ON Escherichia coli