CERTIFICATE

This to certify that the work done by NITHYA.R

( Reg.No.81403204014) to submit the dissertation entitled

ANTIMICROBIAL ACTIVITY OF EUCALYPTUS L.,in

partial

fulfillment of the requirement for the degree of Bachelor of

Technology in INDUSTRIAL BIOTECHNOLOGY to Anna

University is an original work and the same has not

been submitted for any degree.

 HEAD OF THE DEPARMENT

MISSR.HEMAVATHY.BTECH.,
PROJECT GUIDE

EXAMINER’S CERTIFICATE

This dissertion submitted to the Anna University in

partial

fulfillment of the requirement for the award of the degree of

 Bachelor in Technology in Industrial Biotechnology
held

in P.R.Engineering College, Tanjore has been valued on

……………

External Examiner
 DECLARATION

I do here by declare that this work has been originally carried

out by me under the guidance and supervision of
MissR.Hemavathy,B-Tech., lecturer, department of industrial
biotechnology, P.R.Engineering College,Tanjore,and this work has
not been submitted else where for any other degree.

Place: Tanjore R.Nithya

Date:

ACKNOWLEDGEMENT

With great pleasure, I sincerely thank a number of people

who have provided an understanding and assistance in this effort,
first & foremost I wish to express my deep sense of gratitude to
the management of our College, Chairman professor
P.Murugesan,M.sc., and
Dr.Y.R.M.Rao,M.E.,Ph.d.,Principal,PREC,for having given me the
opportunity to under go this dissertation work.
 I owe a great deal to my respected project guide
MissR.V.Hemavathy B-Tech.,for the motivation and I express my
sincere thanks and gratitudes to MrsS.ChitraDevi for her
constant encouragement and kind suggestions they had given in
every step through out this dissertation.

I express my sincere thanks to Dr.A.V.P.Karthikeyan

M.sc.,Ph.d., Head of the department of Industrial Bio-Technology,
PREC,for making necessary arrangements for the successful
completion of my dissertation work.

I am thankful to the other staff members Dr.S.Ravikumar,

Ms.N.Hemalatha, Mr.L.D.Naidu, MsR.Ranjani, MsR.JainulArrifa
and the non-teaching staff of Ind bio-tech for their direct,indirect
help and moral support extended towards this dissertation. I would
also like to thank my friends who helped me in this dissertation
work.

Finally with a deep feeling of indebtness, I express my

warm thanks to my parents and well wishers for their large
contribution towards this dissertation.
 Nithya.R

CONTENTS

 INTRODUCTION

 REVIEW OF LITERATURE

 MATERIALS AND METHODS

 RESULT AND DISCUSSION

 REFERENCES

Materials and methods:

Plant systemics

Eucalyptus L.

Family:

Division:
 Class:

Order:

Genus:

Species:

Habitat: India,

Common Names:

Plants leaf collection:

The plant leaf are collected from

Antimicrobial activity

Microbial strains
The methanol extracts (both polar and non-polar subfractions) and
the essential oils were individually tested against a panel of
microorganisms, including , Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa
ATCC 27853,Bacillus subtilis. Bacterial strains were cultured
overnight at 37 °C in Mueller Hinton agar (MHA).

Antimicrobial screening

Two different methods were employed for the determination of

antimicrobial activities; an agar well-diffusion method for the
methanol extracts and disc diffusion method for the essential oils
([NCCLS (National Committee for Clinical Laboratory Standards),
1999]). The MICs of the essential oils against the test
microorganisms were determined by the broth microdilution
method ( [NCCLS (National Committee for Clinical Laboratory
Standards), 1997]). The MICs of amikacin, clindamycine and
ciprofloxacin were also determined in parallel experiments in order
to control the sensitivity of the test microorganisms. All tests were
performed in duplicate.

Agar-well diffusion method

The polar subfractions of the methanol extracts were weighed and

dissolved in phosphate buffer saline (PBS; pH 7.0–7.2), 10 mg/ml;
non-polar ones were dissolved in dimethyl sulphoxide (DMSO), 10
mg/ml. Both subfractions were filter-sterilised using a 0.45- m
membrane filter. Each microorganism was suspended in sterile
saline and diluted at ca. 106 colony-forming units (cfu)/ml. They
were "flood-inoculated" onto the surface of MHA. The wells (eight
mm in diameter) were cut from the agar and 0.06 ml of extract
solution was delivered into them. After incubation for 24 h at
37 °C, all plates were examined for any zones of growth inhibition,
and the diameters of these zones were measured in millimetres.

Preparation of plant extracts:

5gs of powdered leaf materials is soaked separately with

5ml of ethanol, chloroform, methanol, diethylether, acetone.

They were kept for 7 days at room temperature at 31c for

complete extraction. After seven days, the extracts were

filtered through Whatman No.1 filter paper and collected

 separately in bottles and kept in refrigerator.

Micro organisms and culture media:

The microbial species were collected from P.R.E.C, lab.

Only 4 bacterial species were selected for the present study.

Bacterial species:

 Eicherichia coli

 Bacillus subtilis

 Klebsiella aeroginosa

 Pseudomonas aerogenes

Inoculum Preparation:
 Bacterial cultures were sub cultured in liquid medium (LB
broth) at 37°c for 8 hrs and they were used for subsequent tests
(105 - 106 CFU/ml). These suspensions were prepared
immediately before the tests was carried out.

Media Composition:

LB broth LB Agar
Medium

Peptone-5g Peptone-5g

Beef extract-3g Beef extract-3g

Lactose-5g Lactose-5g

Water-1000ml Water-1000ml

Agar-15g

METHODOLGY:
 The following method were adopted to study the anti-
microbial activity of plant extracts.

Disc diffusion method:

1. The plants extract were tested for antimicrobial activity in the

disc diffusion assay.

2. Four important tests strains of bacteria viz Escherichia coli,

Pseudomonas aerogenes, Bacillus subtilis, Klebsiella
aerogenosa.

3. Bacterial cultures were maintained on LB Agar at 4 ºc.

4. These bacterial cultures were diluted using lactose broth and

diluted bacterial cultures (0.2ml) were spread over sterile LB
Agar plates.

5. 0.2 ml of the plant extract was allied per sterile filter paper
disc (Whatmann No.1 6mm in diameter) the discs were
allowed to dry before being placed to the agar plates.
6. Each plate contained four paper discs with plant extract. Each
extract was tested .

7. The plates were incubated at 37 ºc for 24hrs. Then inhibition

zones were recorded.

8. Thus the anti-microbial activities of plant extract were

indicated by clear zones of growth inhibition.
Result and discussion:
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