1. Study of anti-microbial activity of
Hibiscus rosacinensis leaf extract.
-Arijit Chakraborty
Project done under supervision of
Biswanath Ghosh

2. Theory:
Anti-microbrial:
An anti-microbial is a substance that kills
or inhibits the growth of microorganisms[1] such
as bacteria, fungi, or protozoan's. Antimicrobial drugs
either kill microbes (microbiocidal) or prevent the growth
of microbes (microbiostatic). Disinfectants are
antimicrobial substances used on non-living objects or
outside the body.
Main classes:
There are mainly two classes of antimicrobial
drugs: Those obtained from natural sources:
 Beta-lactam antibiotic (such as penicillins, cephalosporins)
Protein synthesis inhibitors (such
as aminoglycosides, macrolides, tetracyclines, chloramph
enicol, polypeptides)

3. • Synthetic agents:
– Sulphonamides, cotrimoxazole, quinolones
– Anti-virals
– Anti-fungals
– Anti-cancer drugs
– Anti-malarials
– Anti-tuberculosis drugs
– Anti-leprotics
• Anti-protozoals

4.  Apparatus required:
i) Round bottom flask (RBF)
ii) Socks let apparatus.
iii) Beaker (1000 ml, 500 ml, 250 ml)
iv) spatula
v) Glass rod
vi) Funnel
 Chemicals required:
i) Ethanol
ii) Chloroform
iii) Pet ether
iv) Phenol
v) Peptone, Nacl, Beef extract
vi) Distilled water

5. Procedure of Solubility chake:
8 test tubes are taken. 4 of them cold
water and rest are hot water.
Procedure of Thin Layer Chromatography:
At first TLC plate are prepared by silica gel
and water and dry it. Then TLC chamber are
prepared by organic materials like acetic
acid, methanol and water (3:2:1).

6. Procedure:
I. Leaves are collected and dried in room
temperature up to one week.
II. Then leaves are crushed in mixing grinder. (Weight
of leaf is 50 gm.)
III. Then the leaves are extract by using socks let
apparatus.
IV. Four types of extraction are performed using
Organic material.
I) Ethanol Extraction.
II) Chloroform Extraction.
III) Pet. Ether Extraction.
IV) Water Extraction.
V. In ethanol extraction ethanol is used 250 ml.
VI. In chloroform extraction chloroform is used 250
ml.
VII. In pet ether extraction pet ether is used 250 ml.

7. VIII. Extraction sample are taken in to petridishs.
IX. Then dried the Petridis.
X. Then chake the solubility of the extract in hot and cold
water.
XI. Then chake the phenol coefficient by thin layer
chomatography (TLC) method.
XII. Than redal walkar coefficient test are performed to
chake the anti microbial activity of Salmonella typhi in
nutrient broth.
XIII. Then chake the result.

8. Result
 Observation table of solubility:
Sample Cold water Hot water
Ethanol extract Soluble Soluble
Chloroform extract Insoluble Insoluble
PET-Ether extract Insoluble Insoluble
Water extract Soluble Soluble

9. Observation table of TLC:
Sample Response
Ethanol extract + ve
Chloroform extract -Ve
PET-Ether extract -Ve
Water extract + ve

10. Observation table of R.W.C:
Concentration Time Of contact (gm)
2(1/2) 5 7(1/2) 10
1/100
(phenol)
+ + - -
1/100 (ethanol) + + - -
1/200
(ethanol)
+ - - -
1/400 (ethanol) + - - -
1/500 (ethanol) + - - -

11. Concentration Time Of Contact (gm)
2(1/2) 5 7(1/2) 10
1/100 (phenol) + + - -
1/100 (water) + + + -
1/200 (water) + + - -
1/400 (water) + - - -
1/500 (water) + - - -

12.  Calculation:
Formula:
Rideal walker coefficient= (MIC of phenol/ MIC of
sample)
Ethanol extraction:
R.W.C = (MIC of phenol/MIC of ethanol)
= {(1/100)/(1/100)}
= 1
Water extraction:
R.W.C = (MIC of phenol/ MIC of water)
= {(1/100)/(1/200)}
= 2

13.  Conclusion:
After performing the experiment, it
was found that Rideal walker coefficient of
ethanol extraction is 1 and water extraction is
2. So, 1/100 dilution of ethanol extraction is
equal to 1/100 dilution of phenol and 1/200
dilution of water extraction is effective as the
1/100 dilution of phenol.