Phytochemical screening and evaluation of the antibacterial

activity of combined plant extracts(Cucurbita maxima, Calotropis

gigantea, Ficus racemosa, Lagenaria siceraria) and green
synthesized silver nanoparticles.

Supervised by:
Dr.Md.Nazmul Hasan
Professor,
Dept. of Genetic Engineering and Presented by:
Biotechnology Md.Mosarrof Hosen
Student Id: Ms 220605
Dept. of Genetic Engineering and
Biotechnology
Index:

• Background
• Objectives
• Materials and methods
• Expected outcomes
• Conclusion
• List of materials
Background:
Used as folk medicine.
Rich in nutrients, antioxidants and may have
other biologically active compounds.(source-
google).
Escalating Antibiotic resistance .
Eco-friendly nature and diverse application
of silver nanoparticles( AgNPs).
 Objectives:
The objectives of this research proposal are-
 To screen the different phytochemicals in qualitative means.
 To evaluate the antibacterial activities of these plant extract individually and
in combination(1:1)
 To green synthesis of sliver nanoparticles and evaluate their antibacterial
activities.

 To demonstrate the lowest amount of antibacterial agent causing microbial

mortality by the MBC test

 To demonstrate the lowest level of antibacterial agent significantly inhibiting

growth by the MIC test.
 Material and Methods
1.plant extract preparation
Plant collection

Washing & drying

Grinding

Powder of the dried part

Macerated with methanol for3 days

(1 gm sample in 10 ml of solvent) (Pandey et al.,
n.d.).

Filtration & evaporation

Crude methanolic extracts

 Materials and Methods cont…
2.Qualitative Phytochemical • Concentrated H SO4
2

screening: • Distilled water

For steroids &
terpenoids

Equipment and reagents: • 5%ferric chloride

• Test tubes • 10% ferric chloride
For tannins

• Micropipette • Distilled water

• Extracts • Concentrated H SO4
2 For phenol

• Distilled water
For saponins
For quinone
Diluted NaOH
Diluted HCL acid For flavonoids

• Chloroform
 Materials and Methods cont…
• 3.evaluation of antibacterial activity: aqueous extracts

Methanolic
extracts
AgNO3
Single extracts Combined extract
0.02M

AgNPs-AE
Antibacterial using
activity AgNO3
0.02M

Antibacteri
al activity
 Materials and Methods cont…
• A)preparation of Mueller Hinton Agar: (100ml)
Composition of Mueller Hinton Agar (MHA)
Ingredients Gms/Litre
Beef extract 2.0
Acid hydrolysate of casein 17.5
Starch 1.5
Agar 17.0

• Final pH 7.3 +/- 0.1 at 25ºC.

As for,
• 1000ml distilled water 38g of Mueller Hinton agar powder is required
• For 100ml distilled water =38*100/1000g
=3.8g of Mueller Hinton agar powder will be
required.
 Materials and Methods cont…
• Heat with frequent agitation and boil for
one minute to completely dissolve the
medium.
• B)preparation of Mueller Hinton
Broth:(50ml) Composition of Mueller Hinton broth (MHB)

As for,
• 1000ml distilled water 21g of Mueller
Hinton broth powder is required
• For 50ml distilled water =21*50/1000g
=1.05g of
Mueller Hinton broth powder will be
required.
 Materials and Methods cont…

• C)sterilization process:
• I ) MHA & MHB , Petri dishes, test tubes and other glass ware will
be sterilize by autoclaving at a temperature of 121ºC and a
pressure of 15-lbs/Sq inch for 15 minutes.

• ii ) Tips, pipette, tissue paper, syringe, and paraffin will be keep in

a laminar hood and UV light will be switch on 15 minutes before
working in a laminar hood to avoid accidental contamination.
 Materials and Methods cont…
• D) preparation of test culture:
• 20ml Mueller Hinton agar media will be pour into each petri dishe.

• Media will be store in the refrigerator at 4º C

• With the help of an inoculating loop, the test organisms will be transfer to
the nutrient broth in an aseptic condition

• The inoculated broth will be then incubate at37ºC for overnight( 16-18h) to
assure the growth of test organism.
 Materials and Methods cont…
• E) Preparation of plant extract solution:

• Different amounts of plant extract (50,100,, and 200 mg) will be put inside an
Eppendorf tube to make different dosages of plant extract solution.

• Each tube will be filled with 1 ml of DMSO.

• The mixture will be uniformly mixed by using a vortex mixture.

• Plant extract solution will be stored in the refrigerator at 4ºC.

 Materials and Methods cont…
i) agar well diffusion assay: 100µl of MHB will be poured on each petri dish

The MHB will be swabbed by using sterilized cotton swab & will be left for drying

A circular 6/8 nm diameter well will be punched aseptically with a sterile cork
bohrer.

100µl of methanolic extracts will be dispensed into the wells

5% DMSO will be dispensed into the well and reference antibiotic discs will be
placed on the surface of the plate as negative and positive control respectively.

Then the plates will be incubated for 24h at 37ºC

 Materials and Methods cont…
• ii)Disk diffusion assay:
100µl of MHB will be poured on each petri dish

The MHB will be swabbed by using sterilized cotton swab & will be left for drying

The test substance of 0.05ml,0.1ml and 0.2ml is then put on filter paper discs
(approximately 6 mm in diameter) and soaked properly

then put on the agar surface.

Reference antibiotic disk will be put on agar surface.

Under ideal circumstances, the Petri dishes will be incubated

 Materials and Methods cont…

• G) Determination of zone of inhibition:

• After 24 h, antibacterial activity was determined by measurement of

diameter zones of inhibition (mm) against the test organisms around each of
the extracts and the antibiotics.

• The culture plates were examined and zone of inhibition measured in

millimeter scale (Dash et al., 2012).
 Materials and Methods cont…
MIC
• MIC stands for minimum inhibitory concentration, which is the lower
concentration
• of an anti-microbial that will inhibit the visible growth of a microorganism
after
• overnight incubation.
1. Preparation of media:
For 30 mL media Preparation:

• Measure 0.63 g of Muller Hinton broth in analytical balance

• Then dissolve it in 30 mL distilled water

 Materials and Methods cont…

• Preparation of resazurin solution:

For 1mL 0.02% (wt./vol) resazurin solution,

Measure 0.2 mg of resazurin powder in an analytical balance

Then dissolve it in 1 ml distilled water

Then vortex the mixture

 Materials and Methods cont…
• Preparation of Bacteria Inoculum:
I. use a loop to touch the top three to five colonies of the same
morphological type from an agar plate culture
II. suspend in 5 mL of sterile Muller Hinton broth
III. Incubate at 370 C in a shaker
IV. Adjust Turbidity of the activity growing cell by McFarland
standard using sterile respective broths to produce a
standardized microbial inoculum of approximately 1.5 ×108
CFU/ml (for Bacteria)

• The standardized microorganism suspension was then diluted by

1:100 in MHB broth.
 Materials and Methods cont…
• Extract Preparation:
For 64 mg/mL extract,

Measure 64 mg extract in an analytical balance

Then add 1 mL of DMSO

For 32 mg/mL= Take 100 μL from 64 mg/mL +50 μL normal saline+50 μL MHB

For 16 mg/mL= Take 100 μL from 32 mg/mL +50 μL normal saline+50 μL MHB

For 8 mg/mL= Take 100 μL from 16 mg/mL +50 μL normal saline+50 μL MHB
 Materials and Methods cont…
For 4 mg/mL= Take 100 μL from 8 mg/mL +50 μL normal saline+50 μL MHB

For 2 mg/mL= Take 100 μL from 4 mg/mL +50 μL normal saline+50 μL MHB

For 1 mg/mL= Take 100 μL from 2 mg/mL +50 μL normal saline+50 μL MHB

For 0.50 mg/mL= Take 100 μL from 1 mg/mL +50 μL normal saline+50 μL
MHB

For 0.25mg/mL= Take 100 μL from 0.50 mg/mL +50 μL normal saline+50 μL
MHB
 Materials and Methods cont…
Working Procedure:
1. Add a volume of 50μL of test material in the first row of the plate.
2. Add 50μL MHB to each well for serial dilution.
3. Serial dilutions were performed using a micropipette such that
each well had a total volume of 50 μL of the test material in
descending concentration.
4. Add 50 μL bacterial suspension to each well.
5. The plate is placed at 37 0 c for 18-24 hr.
6. After incubation, Add 30 μL of Rasuzurin indicator solution in
each well.
7. Observe color changes after 2-4 hrs. after indicator addition.
 Materials and Methods cont…
• Result:
• • Any color changes from purple to pink to colorless indicated the growth of microbes
• • The lowest con. At which no color change occurred was taken at MIC value of exact

MBC Protocol
MBC stands for the minimum bacterial concentration, the lowest concentration of
the microbial that will prevent the growth of an organism after sub-culture into
antibiotic-free media.
• Preparation of media:
• For 30 mL media Preparation:
• Measure 1.14 g of Muller Hinton Agar in analytical balance
• Then dissolve it in 30 mL distilled water
 Materials and Methods cont…
• Working Procedure:
• Take 10μL of MIC; MIC (upper value 1), MIC (Upper value 2)
• Spread it on the MHA plate for further growth of bacteria
• Incubate the plate at 370c for 24 Hours
• Result:
• Select the MBC value by counting <10 colonies of respective Bacteria.
• Repeated this procedure three times.

• Ref:
• 1. Resazurin-based 96-well plate microdilution method for the determination of
minimum inhibitory concentration of biosurfactants
• 2. Antibacterial activities of the extracts, fractions and isolated compounds from
cancarium patentinervium Miq. Against bacterial clinical isolates.(BMC journal)
 Materials and Methods cont…

• Green synthesis of silver nanoparticles:

Aqueous extract preparation:
1. Take 10mg of leaf extract in conical flask
2. Add distilled water up to 100ml
3. Boil and stir at 80-90ºC
4. Cool down at room temperature
5. Filter the extract using whatman no 1 filter paper
 Materials and Methods cont…
• Preparation of 0.02M silver nitrate solution:
1. Weight 1.7g silver nitrate powder in conical flask
2. Add distilled water up to 500ml
3. Gently shake at room temperature
Working procedure:
Mix aqueous extract with silver nitrate solution in the ration of (9:1)
Incubate it in dark condition at room temperature for 24h
Measure the absorbance at 300-600nm
Centrifuge at 6000rpm for 20 min twice and powder will be obtained followed
by washing three times with in d.w
Dried it at 60-80ºC and sealed it in a tube.
 Expected outcome
Presence of various phytochemicals.
May have noticeable antibacterial agent
Better result of plant extract in combined form.
Anticipates the development of silver nanoparticle with
enhanced biological activities.
This findings may provide insights into the potential use of
these plants and nanoparticles in medicine and related
fields.
List of chemicals
Thank you.