Title: Control of microbial growth

Objective:

1. To learn the disk diffusion technique in controlling a microbial growth.

2. To examine the changing in inhibition zone with the change of the
concentration of the ampicillin.

Introduction:

When a filter paper disc impregnated with a chemical is placed on agar

the chemical will diffuse from the disc into the agar. This diffusion will place the
chemical in the agar only around the disc. The solubility of the chemical and its
molecular size will determine the size of the area of chemical infiltration around
the disc. If an organism is placed on the agar it will not grow in the area around
the disc if it is susceptible to the chemical. This area of no growth around the
disc is known as a “zone of inhibition”.
The control of microbial growth is necessary in many practical situations,
and significant advances in agriculture, medicine, and food science have been
made through study of this area of microbiology. Control of microbial growth
means to inhibit or prevent growth of microorganisms. This control is affected in
two basic ways such as by killing microorganisms or by inhibiting the growth of
microorganisms. Control of growth usually involves the use of physical or
chemical agents which either kill or prevent the growth of microorganisms.
Agents which kill cells are called cidal agents where agents which inhibit the
growth of cells or without killing and it referred to as static agents. Thus, the
term bactericidal refers to killing bacteria, and bacteriostatic refers to inhibiting
the growth of bacterial cells. A bactericide kills bacteria
Antibiotics are one of antimicrobial agents used in vivo. Antibiotics are
microbially produced antimicrobial agents. Antibiotics, produced by many
different bacteria and fungi, apparently have the sole function of inhibiting or
killing other microorganisms. Ampicillin which is semisynthetic penicillin is
classified in a group of β-lactam antibiotics. All β-lactam antibiotics share a
characteristic structural component, the β-lactam ring.
Materials:
Sterilize forceps 1
Sterilized cotton swab 2
Bacteria broth culture
LB plate 2
Micropipette P20 and P200
Microfuge tubes 5
Antibiotic (Ampicillin) 200 µl (50 mg/ml)
Distilled water
Sterilized disks 10 pieces
Sterilized yellow tips 10 tips
Marker pen for labeling

Procedure:

1. A suspension of bacteria culture at OD600 0.6 were given.

2. The organism is inoculated onto the LB agar plate by using a sterile cotton
swab. Streaking is done in three directions over the entire agar surface. The
organism grows rapidly on Luria-Bertani (LB) agar.
3. The plate is allowed to dry for five minutes.
4. While waiting for the plate to dry, the antibiotics are diluted to 10, 100, and
1000 times.
5. By using a sterilized forceps, the disks are placed with an equal distance
apart from each other and four disks are put on the 100 mm diameter plate.
6. A 50 µl of different dilution of the antibiotics is pipette onto the disk and it is
incubated about 5-10 minutes at room temperature followed by 16-20 hours
at 37oC.
7. The step 6 is repeated with 40 µl of different dilution of the antibiotics.
8. The diameter of the inhibition zone is measured.
Discussion

The technique that use in this practical is disk diffusion technique. There
have five side in the plate which 10 -1 dilution factor of ampicillin, 10-2 dilution
factor of ampicillin, 10-3 dilution factor of ampicillin, original ampicillin (pure
ampicillin) and the control where that is the distilled water. A zone of inhibition is
created with a diameter proportional to the concentration of ampicillin. That why
the concentration are effect the diameter of inhibition zone. The measurement
diameter of inhibition zone in this experiment show that the different result base
on the dilution factor of ampicillin that applied. There have several parts that
importance in this practical such as the inhibition zone, the application of paper
disk on agar plate and also factor of dilution of ampicillin. Inhibition zone is the
area around a paper disk or colony of E.coli where no other organisms are
growing. The antibiotic then diffuses into the agar away from the disk. If the
bacteria are sensitive to the antibiotic, they will not grow near the disk.
The size of the zone is proportional to how sensitive the organism is. If the
organism is resistant to the antibiotic, it will grow right up to the disk. The
application of paper disk is as a plat form for the ampicillin in the plat that
cultural by E.coli. The paper disk as the reference point after the put in the plate,
so that the end of the experiment show that the disk in the canter of the
inhibition zone and it make easier to measure that area (inhibition zone). The
dilution factor of ampicillin is the manipulation that that need to test in this
practical where the concentration of ampicillin will give the effect the diameter of
inhibition zone, as result show that the pure ampicillin is about 3.33 cm
diameter. Because of the dilution factor the diameter of became decreasing
follow the factor of dilution.
In this practical, there have several procedures that should be careful and
all the work need to in the lamina flow because the plate is very sensitive to the
other bacteria in air. Antiseptic procedure also importance during handle this
part because e.cli also as a pathogenic bacteria. The antiseptic technique like
wash the glove with ethanol, flame the loop and many more should be apply in
this experiment. It is because to reduce the risk of the samples are
contamination and get the good result after 24 hour. The sample should keep in
oven with 37OC because that is the optimum temperature that E.coli can growth
fast suitable to the growth part of ampicillin.
As the result of this experiment there is show that the concentration of
ampicillin causes of the dilution factor that applied give result with different
diameter.