ANTI-BACTERIAL AND ANTI-FUNGAL ACTIVITY

MECHANISM AND RESULT INTERPRETATTION

AYESHA SADIQA
2015-PhD-CHEMISTRY-03
SUPERVISOR
PROF. DR. SYEDA RUBINA GILANI
DEPARTMENT OF CHEMISTRY
UNIVERSITY OF ENGINEERING AND
TECHNOLOGY , LAHORE
 INTRODUCTION
Antibiotics are one of our most important weapons in
fighting bacterial infections and have greatly benefited
the health-related quality of human life since their
introduction. These health benefits are under threat as
many commonly used antibiotics have become less and
less effective against certain illnesses not, only because
many of them produce toxic reactions, but also due to
emergence of drug-resistant bacteria. it is essential to
investigate newer drugs with lesser resistance.
Antibiotics function by preventing the cell division of
microorganism, inhibiting DNA. Unfortunately, the commonly
used antibiotics have very low MIC as a result the dosage of
antibiotics have to be increased and the treatment can prolong
from 6 weeks to months. this prolong period leads to toxicity of
antibiotic and mutation in DNA of microorganism to develop a
resistant bacterial strain. Thus there is a crucial need to
improve their concentration in order to reduce the toxic effects
of various drugs on human body.
Materials and Methods:
Following Agar medias, instruments and apparatus is required for
Antimicrobial studies
Nutrient Agar
Nutrient Broth
Potato dextrose Agar, Sabouraud Dextrose Agar
Laminar flow hood
Cool incubator and Dry air incubator
Autoclave
Boiling water bath
pH Meter
 STERILIZATION

The process of removal of all microorganisms and other

pathogens from an object or surface by treating it with
chemicals or subjecting it to high heat or radiation.

Sterilization also refers to procedures that result in infertility.

Sterilization can be achieved with one or more of the

following: heat, chemicals, radiation, high pressure.
STEAM STERILIZATION OR AUTOCLAVING:
Liquids, culture media (broth and agar) are autoclaved at 15 psi
and 121 oC for 15 minutes in autoclave. all liquids should be
covered with aluminium foil. this involves high pressure and
temperature
DRY HEAT:
All glass apparatus is wrapped in brown paper and is heated at
a temperature of 180-200 oC in hot air oven for 20 minutes.

FLAMING:
The loops and forceps are directly heated on flame to red hot.
(natural gas flame, sprit flame)

GLASS BEAD STERILIZATION:

Glass beads are heated to 250 oC and then all glassware is
placed on them which by heat sterilize.
STERILIZATION BY CHEMICALS:
Ethylene oxide, ozone, formaldehyde, sprit, hydrogen peroxide,
nitrogen peroxide etc. are used for chemical sterilization.
STERILIZATION BY RADIATIONS:
Electromagnetic radiations, gamma radiation and ultra violet
radiations are employed to sterilize.
ANTIBACTERIAL ACTIVITY:
Nutrient agar is a matter composed of peptone 5g, beef extract 3g
and sodium chloride 8g used to grow the bacterial culture on petri dish
or in test tube in form of slant and nutrient broth is composed of
peptone 5g, beef extract 1g, saodium chloride 5g and yeast extract
2g used to grow bacteria and is called inoculum, which is used for
antimicrobial activity.
ANTIFUNGAL ACTIVITY:
For antifungal activity dextrose potato agar or sabouraud dextrose agar is used.

Broth and Agar preparation:

The quantity of agar is mentioned on different manufacturer’s brand bottle. Weigh that
quantity in respective amount of water and mix it thoroughly so that there are no
lumps. Then heat the vessel containing agar in boiling water bath for 30-45 minutes to
get a transparent media. After 45 minutes remove the vessel from boiling water bath
and cool it enough so that it does not solidify. Maintain the pH according to the label
with acid or base. After maintaining the pH cover the vessel with aluminium foil and
place it in autoclave for Sterilization at 15 psi and 121 oC for 20 minutes.
LAMINAR FLOW HOOD:
Laminar flow hood is a safety cabinet used to perform
antibacterial and antifungal activity in sterilized atmosphere.
it is provided with blower which contain different kinds of
filters and UV light. Prior to use the UVlight is turned on for
15-20 minutes and laminar flow’s window and inner side is
cleaned with formaldehyde or sprit. before starting work UV
light is powered off and blower is turned on.
Media Plates Preparation:
The prepared media and cooled glass ware is kept in laminar
flow hood and there they are uncovered.
Semi-hot agar media is spread on petri dishes (approx. 20ml
or depending on the diameter of petri dishes) and keep them
still for complete settling of agar in petri dishes. on settling
cover the petri dishes and place them in incubator at 37 oC for
24 hours. This is done to check any contamination of
pathogens. If it will be clear this means no contamination.
BACTERIAL CULTURE GROWTH:

There must be pure bacterial strains either purchased from market or

isolated from different sources. Each bacterium strain and fungus mould
has a specific ATCC number
Pour 10-15ml autoclaved nutrient broth in sterilized test tubes. take
platinum loop and heat it red hot to sterilize it and take some strains
from pure and transfer them to test tube containing nutrient broth and
cover it with sterilized cotton plug. In the same way again heat the loop
to red hot and take some strains and transfer them to either petri dish
or test tube containing nutrient agar. and place them in incubator for 24
hours at 37 oC.
Antibacterial Activity:

Different procedures are adopted for anti-bacterial activity such as paper disc method, well diffusion
method and spectrophotometric method.

After bacterial and fungus growth antibacterial and anti-fungus activity id checked. The prepared agar
plates and bacteria containing nutrient broth test tubes are kept in laminar flow. Uncover the petri dish and
pour some broth on it and spread it on whole petri dish and discard the extra. Now soluble the solid samples
in a solvent in which it gives maximum solubility. If using paper disc method dip the sterilized paper discs
in sample and place them on the petri dish containing bacterial culture. In case of well diffusion method
make a well with the end of yellow tip and dispense 5µl sample in well with the help of micropipette.

Place the petri dishes in incubator for 24 hours at 37 oC. After 24 hours measure the zone of inhibition with
the help of vernier caliper or measuring scale in mm or cm. Solvent and controls must also be used. If the
zone of inhibition is greater than solvent or control then the sample will be active against bacteria.
ANTIFUNGAL ACTIVITY:
For anti-fungal activity the semi hot agar is poured onto the petri dishes
and 200-250µl sample is dispensed with micropipette in the same petri
dish and mixed thoroughly and keep it still for settling. on settling place
spores of fungus in the centre of petri dish containing the sample. Anti-
fungal control and solvent must also be run and place the petri dishes in
incubator for specific time and temperature depending on the fungi.
After required time period measure the zone of inhibition with the help
of vernier caliper or measuring scale in mm and cm.
if the zone of inhibition is lesser than the controls then sample have a
good activity against fungus.
MECHANISM:
The suggested mechanism of antibacterial activity is that the metal ions in a
complex form strong bonds with the functional groups (carboxylic, imidazole,
thiol and amine) of the proteins present in the cell membrane of the bacterium.
As the membranes of bacteria come in contact with the metal ions, serious
damage occur to them. The nutrients, and other essential components of the
cytoplasm leaks out of the cell which results in microorganism’s death. The
structural changes in membrane causes increased permeability as a result the
transport system of the cell collapsed and the microorganism dies. Metal ions
not only damage the cell membrane buts also inhibit the activity of several
enzymes. The antibacterial activity depends on three things, the concentration
of metal, the way the metal interacts with bacteria and the different cell
membrane structure of gram positive and gam negative bacteria.
THANKS