Micro practical

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Antimicrobial
sensitivity tests

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Antimicrobial sensitivity test: It is a test which measure
the ability of an antibiotic or other antimicrobial agent to
inhibit bacterial growth.
Antibiotics: substance or biochemical compound
produced by microorganism that kills, or inhibits the
growth of bacteria.
ir Alexander Fleming (1881 –
1955): observed inhibition of
staphylococci on an agar plate
contaminated by a Penicillium
mold

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Bactericidal: antibiotics kill bacteria, examples:
Penicillins, Cephalosporins, Cotrimoxazole….
Bacteriostatic: inhibition growth of bacteria, examples :
Chloramphenicol , Tetracyclines, Erythromycin……

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Classification of antibiotics according to mode of action:
A. Inhibitors of cell wall synthesis:
Penicillins.
B. Disrupters of cell membranes:
Polymyxins.
C. Inhibitors of protein synthesis:
Tetracyclines.
D. Inhibitors of nucleic acid synthesis:
Rifampin.

5. Classification According to Mode of Action:
DNA
Ribosome
Inhibit protein
synthesis

6. Antimicrobial sensitivity test
1. Disk diffusion method: (Kirby-Bauer Method)
2. Epsilometer method (E Test)
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1- Disk diffusion method (Kirby-Bauer Method)
 It is a simple procedure to determine the sensitivity of
microorganism to an antibiotic. Mueller-Hinton Agar
media is a special media for this test.

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*Procedure
1.Prepare a pure culture (18-24 hrs): By loop, transfer (2-
4)colonies of the bacteria which will be tested, to the
tube of sterile peptone water (5ml).
2. Prepare a plate of (Mueller-Hinton Agar) media
(M.H.A) (a special media for this test).
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3. Inoculate the plate of (MHA) by dipping a
sterile swab in to the tube of peptone
water (which contain the bacteria ),and
streak the swab over all the surface of the
(MHA) medium.
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4. Place the antibiotic disks on the plate by using sterile
forceps.
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11. 5. Leave the lid agar for (3-5) minutes to allow plate
to dry, & then incubate at 37 ˚C for 16-18 hrs,
inverted incubate plates in incubator.
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12. 6. After incubation, measure the inhibition zone that is formed around
each disc. Measure the diameter of each zone & compare the
results with the standard measured zones tables of CLSI (Clinical
and Laboratory Standard Institute) & are reported as:
1. Sensitive (S)
2. Intermediate (I)
3. Resistant (R )
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Sensitive (S)
Intermediate (I)
Resistant (R )

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16. 2- Epsilometer method E- test
The E test or Epsilometer method was used to show the lowest
concentration of antimicrobial agent that will inhibit the growth of a
particular microorganisms.
The term “epsilometer” refers to a thin, 5 × 50 mm, inert, nonporous
strip with a continuous exponential gradient of antimicrobial agent.
After Mueller-Hinton agar plate is inoculated with a bacterium (as in
the Kirby-Bauer method), the strip is placed, antibiotic side down on the
agar surface. During incubation, the antibiotic will diffuse into the agar
(higher concentrations traveling farther than lower concentrations) and
an elliptical zone of inhibition will develop. The point at which the
inhibition zone intersects the scale printed on the strip is the Minimum
Inhibitory Concentration (MIC).

17. E- test strip