CLB10603 Lab Manual / Exp.

SECTION OF BIO-ENGINEERING
TECHNOLOGY, UniKL MICET.

Experiment 8 : Determination of antimicrobial activity

Objective :

Some microorganisms produce antimicrobial compounds and these

compounds are detected based on their ability to inhibit the growth of
other microorganisms
To perform methods those are normally used to determine the
antimicrobial activity of microorganism.

Overview :

Antimicrobial compounds are chemicals acting against bacteria. They include

disinfectants which show a low selectivity and are only used for surface
decontamination of non-biological material and chemotherapeutics and
antibiotics which have low or minimal activity against higher eukaryotes.

In contrast to chemotherapeutics which by definition are compounds which have

been chemically synthesized in a laboratory, antibiotics are substances which are
produced by certain taxonomic groups of microorganisms and therefore do
occur naturally.

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.

ndh/dbt/July 2010 1
CLB10603 Lab Manual / Exp. 8

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.

Measuring antimicrobial activity

Determining the antimicrobial activity of a compound is often essential for its

effective use. For example, to fight a bacterial infection a series of antibiotics and
antimicrobials will be tested to determine which should be prescribed as a
treatment. In a food processing plant the concentration of disinfectant to use for
cleaning equipment can be determined experimentally to maximize killing
power, yet minimize the amount of antimicrobial used.

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.

A standard assay often used to gauge the effectiveness of an antimicrobial is the

minimum inhibitory concentration (MIC) test. The MIC is the lowest
concentration of a compound that still inhibits its growth. The MIC of a given
compound for a certain bacterial species is determined using a series of test tubes
containing medium in which the microbe will normally grow, but where each
tube contains progressively lower concentrations of the test compound. Each
tube is inoculated with the microbe and after incubation the tubes in which
growth does not occur are noted. The lowest concentration of the antimicrobial
compound that prevents growth defines the MIC. Since many parameters affect
MIC, it is only valid for the specific conditions tested. If it is desired to compare
the effectiveness of different antimicrobials on a certain strain or to examine the
resistance of a number of microbes to a single agent, test conditions need to be
rigorously standardized. In some situations it is important to determine the
minimum concentration at which a compound is lethal to a microorganism and
this is defined as the minimal lethal concentration (MLC). Antimicrobials that
are cidal will normally kill a microorganism at two to four times their inhibitory
concentration, while a static agent will require a much higher concentration and
may not ever be lethal.

ndh/dbt/July 2010 2
CLB10603 Lab Manual / Exp. 8

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.

ndh/dbt/July 2010 3
CLB10603 Lab Manual / Exp. 8

ndh/dbt/July 2010 4
CLB10603 Lab Manual / Exp. 8

Glassware / apparatus and chemicals for 2 student :

1. A bottle of cell suspension or cell broth of Escherichia coli and Bacillus

subtilis
2. 3 Petri dishes contain sterile nutrient agar
3. Sterile nutrient broth to be diluted with 1 % (w/v) of chloramphenicol
4. 2 pieces of Whatman filter paper which is sterilized prior to use (6mm
disc)
5. 1 % (w/v) of chloramphenicol
6. A bottle containing 200 ml of sterile distilled water
7. 10 sterile Universal bottles
8. Sterile forceps
9. Hockey stick
10. 70% ethanol solution

Experimental procedures

A. Disc agar diffusion technique

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

ndh/dbt/July 2010 5
CLB10603 Lab Manual / Exp. 8

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.

B. Minimum inhibitory concentration, MIC (Tube dilution technique)

1. Prepare a serial double dilution of the 1 % chloramphenicol using the

nutrient broth. The final concentration of the antibiotic will now be 0.5,
0.25, 0.125, 0.0625%.
2. Prepare the antibiotic in 2 sets, one for Escherichia coli and the other for
Bacillus subtilis.
3. To each tube of known concentration of the antibiotic, add 0.1 ml of the
cell suspension of the test organisms and vortex them gently.
4. A nutrient broth without any addition of the antibiotic is used as control
5. Incubate all the tubes at 37°C for 24 h.
6. After 24 h, observe the turbidity, and check the minimum concentration
of the antibiotic that prevents the growth of the test organisms.

Reports.

1. Determine the area of growth inhibition of the test organisms by the

agar diffusion technique.
2. Determine the MIC value of chloramphenicol for Escherichia coli and
Bacillus subtilis.
3. Discuss the effectiveness of the methods used.

ndh/dbt/July 2010 6