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Experiment 8 Determination of Antimicrobial Activity
Experiment 8 Determination of Antimicrobial Activity
Experiment 8 Determination of Antimicrobial Activity
SECTION OF BIO-ENGINEERING
TECHNOLOGY, UniKL MICET.
Objective :
Overview :
This phenomenon was first observed by Alexander Fleming (1928) who had
obtained his now famous Penicillium contamination on a staphylococci plate
which he correctly recognized as caused by the synthesis of an antibacterial
compound by the fungus. He however never purified the active ingredient and
also failed to recognize its therapeutic potential. This was left to a research group
in Oxford consisting of Howard Florey and Ernest Chain who demonstrated the
selectivity of Penicillins in 1940. After their emigration to the US during the war
they then developed the large scale fermentation processes in order to satisfy the
growing demand caused by the high casualties suffered during the second WW.
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The late 1940s and early 1950s then saw the discovery of other antibacterial drugs
such as Streptomycin, Chloramphenicol, Neomycin and Tetracyclin. These
compounds were now called antibiotics because they were of biological origin as
opposed to the chemotherapeutics of the early 20th century; these compounds
can act either bacteriocidal or bacteriostatic.
A good antimicrobial assay will achieve two purposes, it first verifies that the
compound actually has the desired antimicrobial activity and second it indicates
the concentration of the antimicrobial that will be needed to inhibit the target
organism. To determine the activity of an antimicrobial compound against
microorganisms, it is tested under carefully controlled conditions. The
effectiveness of a compound is dependent upon a number of conditions such as
the nature of the target microbes, concentration of the microbes, composition of
the environment, time in contact with the compound, temperature, pH and
amount of aeration.
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Another commonly used assay to assess the potency of an agent is the agar
diffusion method. A microbial culture is spread evenly on the top of an agar
plate containing medium that will support its growth. Disks impregnated with
antimicrobial compounds are then placed onto the agar and the plate incubated
at an appropriate temperature for that microbe. During incubation the
antimicrobial compound diffuses away from the disk and into the agar creating a
concentration gradient that is highest near the disk and decreases as one moves
away from the disk. If a microbe is inhibited by the agent, it will be unable to
grow near the disk, which we see as a zone of clearing in the lawn of growth.
Farther away from the disk, where the concentration of the antimicrobial
compound is much lower, growth will be evident. The size of the zone of
clearing around the disk is an indication of the potency of the antimicrobial for
the tested microbe. Using the agar diffusion method it is possible to test a
number of different compounds simultaneously on the same agar plate. Unlike
the previous method, this one does not readily provide the MIC, since we cannot
easily know the concentration at different places in the agar. The agar diffusion
method is most useful in determining the approximate antibiotic sensitivity of a
microbe.
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Experimental procedures
1. Place the agar plates right side up and divide one of the plates into two
sections by scoring the underside of the plate with a marker pen.
2. Label each section on the plate with the name of organism to be inoculated
(Escherichia coli and Bacillus subtilis). – To see the inhibition growth between
the two microbes.
3. Label the other two plates with the name of organism to be inoculated (one
plate for Escherichia coli and one plate for Bacillus subtilis).- to see the
antimicrobial activity of the microorganism
4. Using the cultures from Escherichia coli and Bacillus subtilis, perform the
spread plate method as described in Experiment 2 on the plate which has
been divide into two sections (each organism on each section as being
labeled respectively). These will be used as the test organisms.
5. Perform the spread plate method as well on the other two plates (each
organism on each plate as being labeled respectively).
6. Prepare the filter paper disc of diameter 6 mm using the 2-layer Whatman
filter papers using the paper hole puncher. Sterile the paper discs by
autoclaving at 121°C for 15 min.
7. Hold the filter paper disc using a pair of sterile forceps and dip the paper in
the 1% chloramphenicol solution. Similarly, using the filter paper, dip in the
distilled water which will be used as control.
8. Place the disc on the surface of the agar containing the cultures of E.coli and
B. subtilis. Gently press each disc down with the wooden end of a cotton
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swab or sterile forceps to ensure that the discs adhere to the surface of the
agar. Do not press the discs into the agar.
9. Incubate the plates at 35°C for 24 h and observe the growth inhibition zone.
Reports.
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