Project Report

UNIVERSITY INSTITIUTE OF ENGINEERING AND
TECHNOLOGY
PANJAB UNIVERSITY, CHANDIGARH
STUDY ON THE ANTIMICROBIAL ACTIVITIES OF THE
PLANT EXTRACTS.
Submitted by:Submitted to:
UE111007 Faculty
UE111013 Biotechnology Branch
UE111027 UIET, PU
UM111005

DECLARATION
We hereby declare that the project titled “STUDY ON THE ANTIMICROBIAL
ACTIVITIES OF THE PLANT EXTRACTS.” submitted in partial fulfillment of the
requirement for the degree of Bachelors of engineering in Biotechnology in UIET,
Chandigarh is original and embodies the research work done by Anshika Parashar,
Dhaarna Kukreja, Kamaljit Kaur, Jyoti Yadav under the supervision and guidance
of Dr. Jaspreet Kaur, Present Branch Coordinator, Department of Biotechnology,
UIET, Panjab University. No part of this work has been submitted for any other
degree.
We also declare that this project report is the result of my own effort and that it has
not been submitted to any other university for the award of any other degree.
Approved by,
Dr. Jaspreet Kaur
Project Coordinator
(8th
semester-Major Project)
06/05/2015

INDEX
 Acknowledgement
 Introduction
 Review of literature
o Antimicrobial property
o Antimicrobial screening
o Antifungal property
o E.Coli
o Bacillus
o Pseudomonas
o Staphylococcus aureus
o Candida
 Plant extracts
 Material and method
 Observations
 Results and discussion
 Bibliography

ACKNOWLEDGEMENT
For this research project we would like to extend our deepest gratitude to Dr. Jaspreet Kaur, Present
Branch Coordinator and Dr. Sanjeev Puri, Ex Branch Coordinator, Department of Biotechnology,
UIET, Panjab University for giving us the opportunity to be associated and work on a topic of such
critical importance.
We are also grateful to our department’s other professors for their valuable encouragement and
guidance throughout the research study. We want to thank them for allowing us to pursue our
project work in between various labs and classes. We are highly obliged to the faculty of
microbiology department for allowing us to use their ELISA reader equipment.
We are indebted to all the Lab attendants for their cooperation to use the equipments in their labs
and for helping us with their technical expertise.
Finally, we would like to extend our thanks to our fellow classmates for their candid discussions
on the topic .
Last but not the least we would like to express our gratitude to the department for giving us an
opportunity to undertake research work in 8th
semester of our degree.

INTRODUCTION
Even though pharmacological industries have produced a number of new antibiotics in the last
three decades, resistance to these drugs by microorganisms has increased. In general, bacteria have
the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic
agents. Such a fact is cause for concern, because of the number of patients in hospitals who have
suppressed immunity, and due to new bacterial strains, which are multi-resistant. Consequently,
new infections can occur in hospitals resulting in high mortality. For example, being Gram-
negative bacteria, most Pseudomonas spp. are naturally resistant to penicillin and the majority of
related beta-lactam antibiotics, but a number are sensitive to piperacillin, imipenem, ticarcillin,
or ciprofloxacin. Aminoglycosides such as tobramycin, gentamicin, and amikacin are other
choices for therapy.
This ability to thrive in harsh conditions is a result of their hardy cell walls that contain porins.
Their resistance to most antibiotics is attributed to efflux pumps, which pump out some antibiotics
before they are able to act. The emergence of antibiotic-resistant forms of pathogenic S.
aureus (e.g. MRSA and VRSA) is a worldwide problem in clinical medicine.
The problem of microbial resistance is growing and the outlook for the use of antimicrobial drugs
in the future is still uncertain. Therefore, actions must be taken to reduce this problem, for example,
to control the use of antibiotic, develop research to better understand the genetic mechanisms of
resistance, and to continue studies to develop new drugs, either synthetic or natural. The ultimate
goal is to offer appropriate and efficient antimicrobial drugs to the patient. For a long period of
time, plants have been a valuable source of natural products for maintaining human health,
especially in the last decade, with more intensive studies for natural therapies. The use of plant
extracts and phytochemicals, both with known antimicrobial properties, can be of great
significance in therapeutic treatments. In the last few years, a number of studies have been
conducted in different countries to prove such efficiency. Many plants have been used because of
their antimicrobial traits, which are due to compounds synthesized in the secondary metabolism of
the plant. These products are known by their active substances, for example, the phenolic
compounds which are part of the essential oils, as well as in tannin.
In recent years, a large number of synthetic pesticides have been banned in the western world
because of their undesirable attributes such as high and acute toxicity, long degradation period,
accumulation in food chain and an extension of their power to destroy both useful organism and
harmful pests. Due to the aforementioned considerations, necessitate the search for alternative
control measures to reduce the dependence on the synthetic fungicides.
Also, the synthetic antimicrobial drugs like Imidazole, Oflaxacin, aminoglycosides,triazole and
thiazole have lot of side effects.Hence, there is a need to find alternative methods to stop the
growth of pathogenic bacteria. So, we chose this project which exploits the antimicrobial activity
of plants, which could be the next big thing in Biomedical engineering.

ANTIMICROBIAL PROPERTY
An antimicrobial is an agent that kills microorganisms or inhibits their growth. Antimicrobial
medicines can be grouped according to the microorganisms they act primarily against. For
example, antibacterial are used against bacteria and antifungal are used against fungi. They can
also be classified according to their function. Agents that kill microbes are called microbicidal,
while those that merely inhibit their growth are called micro-biostatic.
The main classes of antimicrobial agents are disinfectants ("nonselective antimicrobials" such as
bleach), which kill a wide range of microbes on non-living surfaces to prevent the spread of illness,
antiseptics (which are applied to living tissue and help reduce infection during surgery), and
antibiotics (which destroy microorganisms within the body). The term "antibiotic" originally
described only those formulations derived from living organisms but is now also applied to
synthetic antimicrobials, such as the sulphonamides, or fluoroquinolones. The term also used to
be restricted to antibacterial (and is often used as a synonym for them by medical professionals
and in medical literature), but its context has broadened to include all antimicrobials. Antibacterial
agents can be further subdivided into bactericidal agents, who kill bacteria, and bacteriostatic
agents, which slow down or stall bacterial growth. Antibacterial are used to treat bacterial
infections. The toxicity to humans and other animals from antibacterial is generally considered
low. However, prolonged use of certain antibacterial can decrease the number of gut flora, which
may have a negative impact on health. After prolonged antibacterial use consumption of probiotics
and reasonable eating can help to replace destroyed gut flora. Antibacterial are among the most
commonly used drugs. For example 30% or more patients admitted to hospital are treated with one
or more courses of antibacterial. An antibacterial is a substance that either kills bacteria or slows
their growth. Antifungal are the agents that use drugs for treatment of fungal infections. 5-Chloro-
1,3-benzoxazol-2(3 H)-one (5-Chloro Benzoxazolinone) contains an azole ring structure. Numbers
of azole compounds are reported as antibacterial and antifungal agents. Benzoxazolinones
naturally occur in plants. They play a role as defense compounds against bacteria, fungi, and
insects.
Bacterial infections are among the important infectious diseases. Hence, over 50 years of extensive
researches have been launched for achieving new antimicrobial medicines isolated from different
sources. Despite progress in development of antibacterial agents, there are still special needs to
find new antibacterial agents due to development of multidrug resistant bacteria he outburst of
drug resistant microbial strains necessitates the studies for synergistic effects of antibiotics in
combination with plant’s derivatives to develop the antimicrobial cocktail with a wider spectrum
of activity and reduction of adverse side effects of antimicrobial agents. Staphylococcus aureus
resistance to the penicillin group of antibiotics is increasing associated with appearance of adverse
side effects such as hypersensitivity and anaphylactic reactions.

ANTIMICROBIAL SCREENING
The microbiological assay is based upon a comparison of inhibition of growth of micro-organisms
by measured concentrations of test compounds with that produced by known concentration of a
standard antibiotic. Two methods generally employed are turbidometric (tube dilution) method
and cylinder plate (cup-plate) method.
In the turbidometric method, inhibition of growth of microbial culture in a uniform solution of
antibiotic in a fluid medium is measured. It is compared with the synthesized compounds. Here,
the presence or absence of growth is measured. The cylinder plate method depends upon diffusion
of antibiotic from a vertical cylinder through a solidified agar layer in a Petri dish or plate to an
extent such that growth of added microorganisms is prevented entirely in a zone around the
cylinder containing solution of the antibiotics. The cup-plate method is simple and measurement
of inhibition of microorganisms is also easy.
 ANTIFUNGAL PROPERTIES:
Fungi are significant destroyers of foodstuffs and grains during storage, rendering them unfit for
human consumption by retarding their nutritive value and often by producing mycotoxins.
Pathogenic fungi are the main infectious agents’ in plants, causing alterations during
developmental stages including post-harvest. In fruit and vegetables, there is a wide variety of
fungal genera causing quality problems related to aspect, nutritional value, organoleptic
characteristics, and limited shelf life. In addition, in some cases fungi are indirectly responsible
for allergic or toxic disorders among consumers because of the production of mycotoxins or
allergens.
PLANT EXTRACTS
Nine different plants and their parts were collected from botanical garden, Chandigarh on the basis
of their potential medicinal activity. Medicinal Plants Garden/ Herbal Garden has been developed
over 40 acres of land. More than 75 species of medicinal trees and 55 species of medicinal shrubs,
herbs & climbers have already been planted in this section. Tree species like Harar, Bahera, Amla,
Neem, Arjun, Maulsari, Bael, Kathal, Sandalwood, Kachnar, Camphor, Lasura, Gular, Rudraksha,
Jamun, Sita Ashok, Guggal, Putranjeeva etc. have been planted alongwith about 55 varieties of
medicinal herbs like Giloe, Akarkara, Gokhru, Ghritkumari, Ashwagandha, Sarpagandha, Tulsi,
Mehndi, Kalmegh, Bhringraj, Mandukparni, Brahmi, Bansa, Vacha etc.
1. Banana leaves( Musa paradisiaca)
The banana is more than just an exceptionally starch-rich fruit, but has a complex biochemistry,
with pharmacologically active properties. Bananas actually contain the catecholamines dopamine
and norepinephrine, the very same adrenal hormones released in the human body when it

undergoes the typical "fight-or-flight" response. It is believed that the banana plant uses the
biosynthetic pathway for catecholamine when under the stress of attack to fight off infectious
pathogens such as in crown rot disease.
Bananas grow in tropical regions of the world and the humid conditions in the tropics also favor
growth of fungi and bacteria. Any insect that eats off a banana leaf will leave it wounded and prone
to attacks from these pathogens. Nature is all about balance and therefore the banana leaf will try
to defend itself by creating bioactive compounds. The leaves are used by the tribes of Western
Ghats in India for bandaging cuts, blisters and ulcers. There are various types of species in Musa
genus and their pharmacological studies have been studied. Plantain banana (Musa sapientum var.
paradisiaca) has been shown to possess ulcer healing activity in rats. They concluded that wound
healing which could be due to its antioxidant effect and on various wound healing biochemical
parameters present in plantain banana. Methanolic extract of M. sapientum var. paradisiaca
showed antiulcer and mucosal defensive factors in normal and non-insulin dependent diabetes
mellitus rats. They concluded that ulcer protective effect of the extract could be due to its
predominant effect on mucosal glycoprotein, cell proliferation, free radicals and antioxidant
systems. Studies with plantain banana (M. sapientum var. paradisiaca) have indicated its ulcer
protective and healing activities through its predominant effect on various mucosal defensive
factors and they concluded that its antioxidant activity may be involved in its ulcer protective
activity. The flower and stem region of banana plant are known for their anti-ulcer, diuretic, anti-
diabetic and antiseptic properties.
All medicinal plants produce important secondary metabolites like terpenoids, flavonoids,
polyphenols, chlorophylls, betalains etc. Among these, phenolic compounds are considered to be
the chief plant constituent because of its capacity to exhibit antioxidant, anti-cancerous and anti-
inflammatory properties. It is known for many pharmacological activities and reports show that
phenolic compounds mainly contribute to this trait. In the present investigation, quantitative
analysis of phenolic compound was carried out on ethanol (96%), acetone and petroleum ether
extracts of fresh banana leaf . Total phenolic recovery was found to be maximum in petroleum
ether followed by acetone and ethanol. In addition, the leaf extracts were tested for antimicrobial
activities. All the three extracts showed excellent antifungal activities against two pathogenic
fungi, but failed to show any antibacterial activities.
2. Drek(Meliaazedarach)
Parts used: leaves and seed
COMMON NAMES: China berry, Persian lilac, Pride of India, China tree, Pride of China,
Umbrella tree, Umbrella China berry, Indian lilac and Drek.
Description: The plant is a small-to medium-sized deciduous tree, 5 to 15 m tall and 30 to 60 cm
in diameter. It has a spreading, dense and dark green crown. Its bark is dark or reddish brown,
smooth, and becoming fissured. The leaves are alternate. Leaflets have short stalks and are thin,
hairless, dark green on the upper surface and paler underneath. They emit a pungent smell when

crushed. Flowers are purple and fragrant. Fruits or berries are yellow, nearly round, smooth, and
fleshy. They are as hard as stone, containing 4 to 5 black seeds.
Known medicinal uses: Leaves: Leaf extract has insecticidal property (azadira chtin) that repels
insects in clothing. The leaves can also serve as feed for goats. Seed oil: The oil is the most active
medicinal product of the plant. It is used as antiseptic for sores and ulcers that show no tendency
to heal. It is also used for rheumatism and skin diseases such as ringworm and scabies. Internally,
the oil is useful in malaria fever and leprosy.
Pharmacology: Powdered dust of fruit insecticidal, crude extract from wood and bark insecticidal,
oil antibacterial. Alcoholic extract (50% ethanol hydroxide) of leaf anthelmentic, oil with
unspecified extract central nervous system depressant, mild analgesic, depression followed by
stimulation in animals. Alcoholic extract (50% ethanol hydroxide) of stem bark anti-cancerous,
antispasmodic and antiviral.
3. Tun(Toonaciliata)
Parts used: Leaves, roots, flowers, young shoots.
During the last few decades there has been an increasing interest in the study of medicinal plants
and their traditional use in the different parts of the world. One of them which is having numerous
traditional is Toona ciliata commonly known as Toona and Red cedar, belonging to the family
Meliaceae. Toona ciliata leaves are the important source of some aromatic components like
coumarin glycoside, tannins, flavonoids, phenolic compounds, triterpenoid, and steroids. Plant
leaves used traditionally from the ancient time as anti-ulcer activity, analgesic activity, antifungal
activity, antimicrobial, activity, anti-tumor activity, anti-feeding activity, insect-repellent,
insecticidal and antiviral.
Toona ciliata originates from the tropical Asia and tropical Australia, but is now much cultivated
throughout the tropics for its timber and as an ornamental or as away side tree. It is extensively
planted in tropical Africa, particularly in East and Southern Africa, but also locally in West Africa,
Madagascar and Mauritius. It was recorded from Zambia and Zimbabwe as early as the beginning
of 20th century. It has locally become naturalized in Southern Africa.
Botany
Tuna ciliata is deciduous or nearly evergreen, monoecius, medium sized plant that grow up to 25
(-35) m height. It is bole branchless for up to 22 m, up to 50 (-100) cm in diameter, with or without
but tresses at the base. Bark surfaces grayish white to brown, usually fissured, and flanking, inner
bark brown to reddish, fibrous, crown rounded, and spread in. Leaves are alternate, innately
compound with (5-) - 9- 15 pour of leaflets; stripules – absent ; petiole 6-11cmlong. Leaflets
lanceolate to ovatelanceolate, (7-) 9-13 (-16) cm × (2-) 3 - 5(-6) cm, asymmetrical base, acute
oracuminate at apex, entire, glabrous, pinnetely veined. Flowers are unisexual, female–male
similar in appearance, regular. Fruit an ellipsoid to obovoid capsule, 1.5–2.5cm long, pendulas,

smooth, reddish brown, many seeded. Flowers and fruits appears in June to August
Antioxidant Activity:-Reactive oxygen species (ROS) possess a strong oxidizing effect and induce
damageto biological molecules, including proteins, lipids and DNA, with concomitant changes in
their structure and function. The major nutritional antioxidants, vitamin E, vitamin C and β-
carotene, may be beneficial to prevent several chronic disorder considerable interest has arisen in
the possible reinforcement of antioxidant defenses, both for chemoprevention and treatment
purposes
Toona ciliata has been medicinally used as a therapeutic agent for a variety of diseases, as we have
illustrated in this article. Moreover, numerous research works have proven its uses beyond the
medicinal ones in experimental animals. The plants appears to have abroad spectrum of activity
on several ailments. The various parts of the plant have been explored for antioxidant ,analgesic,
anti feedant, antiulcer, antimicrobial and many other activities. It is reported to contain cedrelone,
siderin and triterpenoids, which may be responsible for the different biological activities. Hence,
we can isolate some pure phyto pharmaceuticals which in turn can be used as lead molecules for
synthesizing novel agents having good therapeutic activity
4. Sanchar(Barleriaprionitis)
Also known as the porcupine flower is a species of plants in the family Acanthaceae, native to
India & Sri Lanka. Barleria is an erect, prickly shrub, usually single-stemmed, growing to about
1.5 m tall. The stems and branches are stiff and smooth and light brown to light grey in colour.
The leaves are up to 100 mm long and 40 mm wide, and oval-shaped though narrow at both ends
(ellipsoid). The base of the leaves is protected by three to five sharp, pale coloured spines, 10–20
mm long. The yellow–orange tubular flowers are found bunched tightly together at the top of the
plant, but they also occur singly at the base of leaves. The flowers are 40 mm long and tubular,
with several long protruding stalks (stamens). The seed capsule is oval-shaped and 13–20 mm
long, with a sharp pointed beak. It contains two fairly large, flat seeds, typically 8 mm long by 5
mm wide, covered with matted hairs. Barleria has a central tap root, with lateral roots branching
off in all directions.
It is used for various medicinal purposes in ayurvedic medicine. The juice of the leaves is applied
to feet to prevent maceration and cracking in the monsoon season. It is used to treat fever,
respiratory diseases, toothache, joint pains. A mouthwash made from root tissue is used to relieve
toothache and treat bleeding gums. The leaves are used to healing of wounds and to relieve joint
pains. Extracts of the plant are incorporated into herbal cosmetics and hair products to cure skin
and scalp problems
It has also been utilized for treatment of toothache, catarrhal affections, whooping cough,
inflammations, glandular swellings, urinary infection, jaundice, fever, gastrointestinal disorders
and as diuretic and tonic.

A wide range of phytochemical constituents including balarenone, pipataline prionisides,
barlerinoside, verbascoside, shanzhiside methyl ester, barlerin, acetylbarlerin, lupulinoside,
scutellarein have been isolated from the different parts of this plant. Extracts and isolated
phytochemicals from this plant have been found to posses wide range of pharmacological include
antimicrobial, anthelmintic, antifertility, antioxidant, anti-diabetic, anti-inflammatory, anti-
arthritic, cytoprotective, hepatoprotective, diuretic, anti-diarrhoeal and enzyme inhibitory.
5. Sadabahar(Vincaroseus)
Parts used: pink and white flowers
It is an evergreen sub shrub or herbaceous plant growing 1 m tall. The leaves are oval to oblong,
2.5–9 cm long and 1–3.5 cm broad, glossy green, hairless, with a pale midrib and a short petiole
1–1.8 cm long; they are arranged in opposite pairs. The flowers are white to dark pink with a darker
red centre, with a basal tube 2.5–3 cm long and a corolla 2–5 cm diameter with five petal-like
lobes. The fruit is a pair of follicles 2–4 cm long and 3 mm broad. The blooms of the natural wild
plants are a pale pink with a purple "eye" in their centers. It is native to the Indian Ocean island of
Madagascar. This herb is now common in many tropical and subtropical regions worldwide,
including the southern United States.
The species has long been cultivated for herbal medicine and as an ornamental plant.
In Ayurveda (Indian traditional medicine) the extracts of its roots and shoots, though poisonous,
is used against several diseases. In traditional Chinese medicine, extracts from it have been used
numerous diseases. The substances vinblastine and vincristine extracted from the plant are used in
the treatment of leukemia and Hodgkin's lymphoma.
Extracts of entire dried plant contain many alkaloids of medicinal use. The principal alkaloid is
vinblastine, or vincaleukoblastine (vinblastine sulfate), sold as Velban.
6. Kapurhaldi(Curcuma aromatic)
The wild ginger is one among the 80 members of Zingiberaceae family of plants. The perennial
foliage dies down in late autumn and the rhizomes remain dormant in winter. The inflorescence
appears in early spring from the base of the rhizomes. During summer monsoon season and the
immediately following weeks, the plant grows fast and vigorously.
It is a perennial tuberous herb with annulate aromatic yellow rhizome which is internally orange-
red in colour. Leaves are elliptic or lanceolate-oblong, caudate-acuminate, 30-60 cm long, petioles
as long or even longer, bracts ovate, recurved. Flowers: pink, lip yellow, obovate, deflexed,
subentire or obscurely 3 lobbed. Fruits obtained are dehiscent, globose, 3-valved capsules
Wild turmeric has rhizomes with a peculiar fragrance and attractive deep yellow color. The
rhizomes are often used in cosmetic herbal medicines and as a culinary ingredient in limited
quantities as a food flavor. Leaves are broad and very decorative, elliptic with a leaf stem running
as long to the tip of the blade. A fresh stalk with flowers and leaves, cut to proper size and shape,

can be used as a floral indoor decoration in vase for up to 10 days. It is recognized as a medical
herb with strong antibiotic properties. It is believed to play a role in preventing and curing cancer
in chinese medicine. In an effort to remove cell accumulations such as a tumors, curcuma is often
utilized. There are two species commonly used in cancer therapy that, like ginger, have a spicy
taste. It contains aromatic volatile oils that help to remove excessive lipids from the blood, reduce
aggregation of platelets (sticking of the blood cells to form masses), and reduce inflammation.
They are useful in bruises, sprains, cough, bronchitis etc.
ANTIFUNGAL ACTIVITY
Many skin diseases such as tinea and ringworm caused by dermatophytes exist in tropical and
semitropical areas. In general, these fungi live in the dead, top layer of skin cells in moist areas of
the body, such as between the toes, the groin, and under the breasts. These fungal infections cause
only a minor irritation. Other types of fungal infections could be more serious. They can penetrate
into the cells and cause itching, swelling, blistering and scaling. In some cases, fungal infections
can cause reactions elsewhere in the body. For example, a person may develop a rash on the finger
or hand after coming into contact with an infected foot. The dermatophytes, Trichophyton,
Epidermophyton and Microsporumcanis are commonly involved in such infections. An antifungal
is an agent that that limits or prevents the growth of yeasts and other fungal organisms.Plants
synthesise a vast array of secondary metabolites that are gaining importance for their
biotechnological applications. The antifungal activity of the ethanolic extracts of ten Argentinean
plants used in native medicine is reported. Antifungal assays included radial growth inhibition,
disk and well diffusion assays and growth inhibition by broth dilution tests. The chosen test fungi
were yeasts, micro fungi and wood-rot causing Basidiomycetes.Extracts of Larrea
divaricata, Zuccagnia punctata and Larreacuneifolia displayed remarkable activity in the assays
against the majority of the test fungi. In addition to the former plants, Prosopanche americana also
inhibited yeast growth. Plants produce a great deal of secondary metabolites, many of them with
antifungal activity. Well-known examples of these compounds include flavonoids, phenols and
phenolic glycosides, sulphur compounds and saponins. The use of plants for their antifungal
properties which could be used against the pathogen in the organic farming system becomes an
area of interest for the eco friendly mode of disease management. Considering the vast potentiality
of plant as sources for antimicrobial drugs with reference to antimicrobial agents, a systematic
investigation was undertaken to screen the antifungal activity of medicinal plant species,
Oxaliscorniculata (creeping woodsorrel red and green), Ocimumgratissimum(wild basil) and bulbs
of Acoruscalamus (sweet flag), Zingiber officinale (ginger).In recent years, a large number of
synthetic pesticides have been banned in the western world because of their undesirable attributes
such as high and acute toxicity, long degradation period, accumulation in food chain and an
extension of their power to destroy both useful organism and harmful pests. Due to the
aforementioned considerations, necessitate the search for alternative control measures to reduce
the dependence on the synthetic fungicides.

Bacillus
Morphology
Bacillus (genus Bacillus), any of a group of rod-shaped, gram-positive, aerobic or (under some
conditions) anaerobic bacteria widely found in soil and water. The term bacillus has been applied
in a general sense to all cylindrical or rodlike bacteria. The largest known Bacillus species, B.
megaterium, is about 1.5 μm (micrometres; 1 μm = 10−6 m) across by 4 μm
long. Bacillus frequently occur in chains. The cell wall of Bacillus is a structure on the outside of
the cell that forms the second barrier between the bacterium and the environment, and at the same
time maintains the rod shape and withstands the pressure generated by the cell's turgor. The cell
wall is composed of teichoic and teichuronic acids. B. subtilis is the first bacterium for which the
role of an actin-like cytoskeleton in cell shape determination and peptidoglycan synthesis was
identified, and for which the entire set of peptidoglycan-synthesizing enzymes was localised. The
role of the cytoskeleton in shape generation and maintenance is important.
Pathogenicity
Some types of Bacillus bacteria are harmful to humans, plants, or other organisms. For
example, B. cereus sometimes causes spoilage in canned foods and food poisoning of short
duration. Bacillus food poisoning usually occurs because heat-resistant endospores survive
cooking or pasteurization and then germinate and multiply when the food is inadequately
refrigerated. The symptoms of B. cereus food poisoning are caused by toxins produced in the food
during bacterial growth, principally a necrotizing enterotoxin and potent haemolysins (especially
cereolysin). Phospholipases produced by B. cereusmay act as exacerbating factors by degrading
host cell membranes following exposure of their phospholipid substrates in wounds or other
infections. Emetic food poisoning probably results from the release of emetic factors from specific
foods by bacterial enzymes.Most strains of Bacillus are not pathogenic for humans but may, as
soil organisms, infect humans incidentally. A notable exception is B. anthracis, which
causes anthrax in humans and domestic animals. B. thuringiensis produces a toxin (Bt toxin) that
causes disease in insects.
Why this strain is chosen?
Bacillus endospores are resistant to hostile physical and chemical conditions, but in addition
various Bacillus species have a wide range of physiologic adaptations which enable them to
survive or thrive in harsh environments, ranging from desert sands and hot springs to Arctic soils
and from fresh waters to marine sediments. Because the spores of many Bacillus species are
resistant to heat, radiation, disinfectants, and desiccation, they are difficult to eliminate from
medical and pharmaceutical materials and are a frequent cause of contamination. Bacillus species
are well known in the food industry as spoilage organisms.

Escherichia coli
Morphology
E.coli is a Gram-negative (bacteria which do not retain crystal violet dye), facultative
anaerobic (that makes ATP by aerobic respiration if oxygen is present, but is capable of
switching to fermentation or anaerobic respiration if oxygen is absent) and non
sporulating. Cells are typically rod-shaped, and are about 2.0 micrometers (μm) long and 0.25–
1.0 μm in diameter, with a cell volume of 0.6–0.7 μm3. It can live on a wide variety of
substrates. They are rod shaped and the strains that possess flagella are motile. The flagella
have a peritrichous arrangement. They give positive results for Catalase test and negative for
Oxidase test.Optimal growth of E.coli occurs at 37 °C (98.6 °F), but some laboratory strains
can multiply at temperatures of up to 49 °C.
Pathogenicity
E. coli and other facultative anaerobes constitute about 0.1% of gut flora, and fecal–oral
transmission is the major route through which pathogenic strains of the bacterium cause
disease. It is the leading pathogen causing urinary tract infections and is among the most
common pathogens causing blood stream infections, wounds, otitis media and other
complications in humans. E. coli is also the most common cause of food and water-borne
human diarrhea worldwide and in developing countries, causing many deaths in children under
the age of five years.
The ability for E. coli to exist as a human-adapted commensal compounded with its natural
tendency for frequent genetic exchange, its ubiquitous presence, and the enormous, diverse,
and largely uncharacterized reservoir of genetic variation found within the species' collective
genomes all contribute to the emergence of new pathogenic strains. It is one of the few gram
negative organisms which is pathogenic and adapts according to the host, hence, making it
even more difficult to eliminate. Escherichia coli accounts for 17.3% of clinical infections
requiring hospitalization and is the second most common source of infection
behind Staphylococcus aureus (18.8%). Among outpatient infections, E. coli is the most
common organism (38.6%). Antimicrobial resistance in E. coli has been reported worldwide
and increasing rates of resistance among E. coli is a growing concern in both developed and
developing countries8, 9. A rise in bacterial resistance to antibiotics complicates treatment of
infections. Therefore, we chose this strain.

Pseudomonas
Morphology
Pseudomonas is a genus of Gram-negative, aerobic belonging to the family. They are rod shaped
and have one or more polar flagella that provides motility to the organism. They are aerobic and
are positive on catalyst test and oxidase test. The members of the genus demonstrate a great deal
of metabolic diversity, and consequently are able to colonize a wide range of niches. Their ease of
culture in vitro and availability of an increasing number of Pseudomonas strain genome sequences
has made the genus an excellent focus for scientific research; the best studied species include P.
aeruginosa in its role as an opportunistic human pathogen, the plant pathogen P. syringae, the soil
bacterium P. putida, and the plant growth-promoting P. fluorescens.
Antibiotics used
Aminoglycosides, Gentamycin, Amikacin, Cephalosporins and Ofloxacin are used in case of
Pseudomonas. But, these medications have lot of side effects on humans. Hence, there is a need
to find alternative ways to treat diseases caused by this organism.
Being Gram-negative bacteria, most Pseudomonas spp. are naturally resistant to penicillin and the
majority of related beta-lactam antibiotics, but a number are sensitive
to piperacillin, imipenem, ticarcillin, or ciprofloxacin. Aminoglycosides such as tobramycin,
gentamicin, and amikacin are other choices for therapy.
This ability to thrive in harsh conditions is a result of their hardy cell walls that contain porins.
Their resistance to most antibiotics is attributed to efflux pumps, which pump out some antibiotics
before they are able to act. Pseudomonas aeruginosa is increasingly recognized as an
emerging opportunistic pathogen of clinical relevance. One of its most worrying characteristics is
its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of
multidrug efflux pumps with chromosomally encoded antibiotic resistance genes and the low
permeability of the bacterial cellular envelopes. Besides intrinsic resistance, P. aeruginosa easily
develops acquired resistance either by mutation in chromosomally encoded genes or by
the horizontal gene transfer of antibiotic resistance determinants. Development of multidrug
resistance by P. aeruginosa isolates requires several different genetic events that include
acquisition of different mutations and/or horizontal transfer of antibiotic resistance genes.
Hypermutationfavours the selection of mutation-driven antibiotic resistance in P.
aeruginosa strains producing chronic infections, whereas the clustering of several different
antibiotic resistance genes in integrons favours the concerted acquisition of antibiotic resistance
determinants. Some recent studies have shown phenotypic resistance associated
to biofilm formation or to the emergence of small-colony-variants may be important in the
response of P. aeruginosa populations to antibiotic treatment.

Staphylococcus aureus
Morphology
Staphylococcus aureus is a Gram-positive coccal bacterium that is a member of the Firmicutes,
and is frequently found in the human respiratory tract and on the skin. It is positive for catalase
and nitrate reduction. S. aureus is capable of secreting several exotoxins and various enzymes such
as coagulase.The temperature range for growth of S. aureus is 7–45°C, with an optimum of 37°C.
They form bunches because they divide in two planes as opposed to their close relatives
streptococci which form chains because they divide only in one plane. Colonies formed by S.
aureus are yellow (thus the name aureus, Latin for gold) and grow large on a rich
medium. Staphylococcus aureus and their genus Staphylococci are facultative anaerobes which
means they grow by aerobic respiration or fermentation that produces lactic acid.
Pathogenicity
Staphylococcus aureus is the most common cause of staph infections and is responsible for various
diseases including: mild skin infections (impetigo, folliculitis, etc.), invasive diseases (wound
infections, osteomyelitis, bacteremia with metastatic complications, etc.), and toxin mediated
diseases (food poisoning, toxic shock syndrome or TSS, scaled skin syndrome, etc.). Infections
are preceded by colonization. Common superficial infections include carbuncles, impetigo,
cellulitis, folliculitis. Community-acquired infections include bacteremia, endocarditis,
osteomylitis, pneumonia and wound infections are less common. S. aureus also causes
economically important mastitis in cows, sheep and goats. Disease-associated strains often
promote infections by producing potent protein toxins, and expressing cell-surface proteins
that bind and inactivate antibodies. Some strains of S. aureus are capable of
producing staphyloxanthin — a golden coloured carotenoid pigment. This pigment acts as
a virulence factor, primarily by being a bacterial antioxidant which helps the microbe evade
the reactive oxygen species which the host immune system uses to kill pathogens. The first-line
therapy is most commonly a penicillinase-resistant β-lactam antibiotic.
It is found in the human respiratory tract and on the skin. It is a successful pathogen due to a
combination of nasal carriage and bacterial immuno-evasive strategies. The treatment of choice
for S. aureus infection is penicillin. In most countries, however, penicillin resistance is extremely
common. The emergence of antibiotic-resistant forms of pathogenic S. aureus (e.g. MRSA and
VRSA) is a worldwide problem in clinical medicine. As a pathogen, it is important to understand
the virulence mechanisms of S. aureus especially the Methicillin-resistant Staphylococcus aureus
(MRSA) in order to successfully combat the pathogen. The increasing population of "super
germs" and antibiotic resistant pathogens have increased pressure on researchers to find
alternative, more effective ways of fighting these "super germs."

Candida
Morphology
Candida is a genus of yeasts and is the most common cause of fungal infections worldwide.
Many species are harmless commensals or endosymbionts of hosts including humans; however,
when mucosal barriers are disrupted or the immune system is compromised they can invade and
cause disease. Although often referred to as "dimorphic", C. albicans is in fact polyphenic. When
cultured in standard yeast laboratory medium, C. albicans grows as ovoid "yeast" cells.
However, mild environmental changes in temperature and pH can result in a morphological shift
to pseudohyphal growth. Pseudohyphae share many similarities with yeast cells, but their role
during candidiasis remains unknown. When C. albicans cells are grown in a medium that mimics
the physiological environment of a human host, they grow as "true" hyphae. Its ability to form
hyphae has been proposed as a virulence factor, as these structures are often observed invading
tissue, and strains that are unable to form hyphae are defective in causing infection. Candida
albicans can also form Chlamydospores, the function of which remains unknown.
One of the most important features of the C. albicans genome is the occurrence of numeric and
structural chromosomal rearrangements as means of generating genetic diversity, named
chromosome length polymorphisms (contraction/expansion of repeats),
reciprocal translocations, chromosome deletions and trisomy of individual chromosomes.
Pathogenicity
Candida are almost universal in low numbers on healthy adult skin and albicans is part of the
normal flora of the mucous membranes of the respiratory, gastrointestinal, and female genital
tracts. In the case of skin, the dryness of skin compared to other tissues prevents the growth of
the fungus, but damaged skin or skin in intertriginous regions is more amenable to rapid growth
of fungi.
Overgrowth of several species including albicans can cause superficial infections such as
oropharyngeal candidiasis (thrush) and vulvo vaginal candidiasis (vaginal candidiasis). Oral
candidiasis is common in elderly denture wearers. In otherwise healthy individuals, these
infections can be cured with topical or systemicantifungal medications (commonly over-the-
counter antifungal treatments like miconazole or clotrimazole). In debilitated or immune
compromised patients, or if introduced intravenously, Candidia may become a systemic disease
producing abscess, thrombophlebitis, endocarditis, or infections of the eyes or other
organs. Typically, relatively severe neutropenia is a prerequisite for the Candida to pass through
the defenses of the skin and cause disease in deeper tissues; in such cases, mechanical disruption
of the infected skin sites is typically a factor in the fungal invasion of the deeper tissues.

Antibiotics promote yeast infections, including gastrointestinal Candida overgrowth, and
penetration of the GI mucosa. While women are more susceptible to genital yeast infections,
men can also be infected. Certain factors, such as prolonged antibiotic use, increase the risk
for both men and women. People with diabetes or impaired immune systems, such as those
with HIV, are more susceptible to yeast infections. Just like antibiotics cure bacterial
infections, antifungal medications save lives by curing dangerous fungal infections. And just
like some bacterial infections are resistant to antibiotics, some fungi no longer respond to the
antifungal medications that are designed to cure them. This emerging phenomenon is known
as antifungal resistance, and it’s primarily a concern for invasive infections with the
fungus Candida. Although antibiotic-resistant bacterial infections are a widely-recognized
public health threat, less is known about the effects of antifungal resistance and the burden of
drug-resistant fungal infections. Some types of Candida are becoming increasingly resistant to
first-line and second-line antifungal medications, namely, fluconazole and echinocandins
(anidulafungin, caspofungin, and micafungin).Approximately 7% of all Candida bloodstream
isolates tested at CDC are resistant to fluconazole, most of which are Candida glabrata. The
stable yet substantial rates of fluconazole resistance and the emergence of echinocandin
resistance are concerning because echinocandins are typically used to treat infections caused
by C. glabrata, the species that’s most often associated with fluconazole resistance. For multi-
drug resistant Candida infections (those that are resistant to both fluconazole and an
echinocandin), the few remaining treatment options are expensive and can be toxic for patients
who are already very sick. Some species of fungi are naturally resistant to certain types of
antifungal medications. Other species may be normally susceptible to a particular type of
medication, but develop resistance over time as a result of improper antifungal use—for
example, dosages that are too low or treatment courses that aren’t long enough. Some studies
have indicated that antibacterial medications may also contribute to antifungal resistance; this
could occur for a variety of reasons, one of which is that antibacterials reduce bacteria in the
gut and create favorable conditions for Candida growth.
MATERIAL AND METHODS

Collection of the required part from the selected plants-
1) (Banana) - leaves
2) Barleriaprionitis ( sanchar) – leaves
3) Barleriaprionitis ( sanchar) - flowers
4) Vincarosia ( sadabhar) – leaves
5) Vincarosia ( sadabhar) – flowers
6) Toona ciliate ( tun) – leaves
7) Meliaazedarach ( drek) – seeds
8) Curuma aromatic ( karpurahaldi) – roots
9) Meliaazedarach ( drek) - leaves
Procedure to prepare plant extracts :-
1) Grind the parts of the plants using pastel and mortal or either using mixer
2) After grinding the samples go for ethanol extraction.
Procedure for ethanol extraction:
1. Take 0.5g of the grind sample and mix it with 10ml of 95% ethanol solution.
2. Incubate it at room temperature for 24-48 hours for the proper extraction.
3. Filter the solutions using filter paper.
4. Air dry the solution for the evaporation of the ethanol.
5. Mix the obtain extract in 1ml DMSO solution.
6. Centrifuge at 800rpm for 5 minutes. Take the supernatant.
7. Again filter it using filter paper of .2 micron using filter assembly in laminar
8. The required extract is obtain.
Procedure to check the ant-bacterial activity of the plant:-
1) Prepare 10ml nutrient broth( for E.coli, Bacillus), sabouraud dextrose broth(for Candida),
brain heart infusion broth (for Staphylococcus).
2) Revive the cultures in respective broths for the fresh growth.
3) Take a 96 well plate and add media, culture and extracts according to the table.
4) Incubate the plate at 30°C and 37°C.
5) Check the ELISA plate in the ELISA reader.
6) Analyze the obtained results.
OBSERVATIONS

Control Optical
density
Media (NB)+E.coli 0.931
Media(NB)+Bacillus 0.682
Media (SDB)+Candida 0.672
Media (NB)+Pseudomonas 0.998
Media(BHIB)+Staphylococcus 0.956
Media (NB)+Extract 1(Banana) 0.042
Media (NB)+Extract 2(drek leaves) 0.0385
Media (NB)+Extract 3(Drek flower) 0.040
Media (NB)+Extract 4 (Sadabhar white
flower)
0.042
Media (NB)+Extract 5(Sadabhar pink flower) 0.041
Media (NB)+Extract 6(Sadabhar leaves) 0.039
Media (NB)+Extract7(tun leaves) 0.041
Media (NB)+Extract 8(Karpurahaldi leaves) 0.0405
Media (NB)+Extract 9(Karpurahaldi root) 0.0415
Media (SDB)+Extract 1(Banana) 0.060
Media (SDB)+Extract 2(drek leaves) 0.077
Media (SDB)+Extract 3(Drek flower) 0.053
Media (SDB)+Extract 4 (Sadabhar white
flower)
0.072
Media (SDB)+Extract 5(Sadabhar pink
flower)
0.057
Media (SDB)+Extract 6(Sadabhar leaves) 0.081
Media (SDB)+Extract7(tun leaves) 0.049
Media (SDB)+Extract 8(Karpurahaldi leaves) 0.043
Media (SDB)+Extract 9(Karpurahaldi root) 0.057
Media (BHIB)+Extract 1(Banana) 0.0465
Media (BHIB)+Extract 2(drek leaves) 0.0425
Media (BHIB)+Extract 3(Drek flower) 0.042
Media (BHIB)+Extract 4 (Sadabhar white
flower)
0.046
Media (BHIB)+Extract 5(Sadabhar pink
flower)
0.046
Media (BHIB)+Extract 6(Sadabhar leaves) 0.042
Media (BHIB)+Extract7(tun leaves) 0.0755
Media (BHIB)+Extract 8(Karpurahaldi
leaves)
0.072
Media (BHIB)+Extract 9(Karpurahaldi root) 0.074
The following readings have been obtained after subtracting the control value in each case. The
control values were taken by taking readings of extract + media and media+culture. Then, these

readings were subtracted from the readings of media+culture+extract. The total volume in the
wells is 200 uL that includes 180uL media and 20uL culture. Later the volume of extracts are
progressively varied as 5uL, 10uL, 15uL and 20uL.The final volume was adjusted by adding
media, so that final volume remains the same in each case. Results obtained were as follows:-

RESULT AND DISCUSSION
Extract 1(Banana leaves)
Extract 2(Drek leaves)
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30
O.D(600nm)
volume(uL)
E.coli
Pseudomonas
Bacillus
Candida
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30
O.D(600nm)
Volume(uL)
E.coli
Bacillus
Pseudomonas
Candida

Extract 3(Drek flower)
Extract 4(Sadabhar white flower)
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30
O.D(600nm)
volume (uL)
E.coli
Bacillus
Pseudomaonas
Candida
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30
(OD(600nm)
Volume(uL)
E.coli
Bacillus
Pseudomonas
Candida

Extract 5(Sadabhar pink flower)
Extract 6(Sadabhar leaves)
0
0.2
0.4
0.6
0.8
1
1.2
0 10 20 30
OD(600nm)
volume(uL)
E.coli
Bacillus
Pseudomonas
Candida
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25
OD(600nm)
Volume(uL)
E.coli
Bacillus
Pseudomonas
Candida

Extract 7(Tun leaves)
Extract 8(Karpurahaldi leaves)
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25
OD(600nm)
Volume(uL)
E.coli
Bacillus
Pseudomonas
Candida
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25
OD(600nm)
volume(uL)
E.coli
Bacillus
Pseudomonas
Candida

Effect of extract on Bacillus
0
0.5
1
0 5 10 15 20 25
OD(600nm)
Volume(uL)
banana leaves drek leaves
drek leaves2 sadabhar white flower
0.682-control
0
0.2
0.4
0.6
0.8
0 5 10 15 20 25
OD(600nm)
Volume(uL)
sadabhar pink flower sadabhar leaves
tun leaves karpurahaldi leaves
karpurahaldi roots

Effect of extracts on E.coli
0
0.5
1
0 5 10 15 20 25
OD(600nm)
Volume(uL)
drek flower sadabhar white flower
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25
OD(600nm)
Volume(uL)
karpurahaldi root

Effect of extract on Pseudomonas
0
0.5
1
1.5
0 5 10 15 20 25
OD(600nm)
Volume(uL)
0
0.5
1
1.5
0 5 10 15 20 25
OD(600nm)
volume(uL)
karpurahaldi roots

Effect of extract on Candida
0
0.2
0.4
0.6
0.8
0 5 10 15 20 25
OD(600nm)
Volume(uL)
0
0.2
0.4
0.6
0.8
0 5 10 15 20 25
OD(600nm)
Volume(uL)
0.682-control

Discussion
On getting the positive results of antimicrobial activity of the chosen plant extracts against
staphylococcus aureus, we extended our research on other bacteria and fungi; E.coli,
Bacillus, Pseudomonas, and Candida. There was inhibition of growth of each
microorganism on using our plant extracts. Also, we observed that the inhibition of
microbial growth increased with increase in amount of plant extract.
E.coli- maximum inhibition is obtained in Karpurahaldi roots using 20µl volume.
Bacillus- maximum inhibition is obtained in Drek flowers using 20µl volume.
Pseudomonas- maximum inhibition is obtained in Sadabhar pink flower using 20µl
volume.
Candida - maximum inhibition is obtained in Tun leaves using 20µl volume.
BIBLIOGRAPHY
 ANTARA SEN
*
AND AMLA BATRA ; EVALUATION OF ANTIMICROBIAL
ACTIVITY OF DIFFERENT SOLVENT EXTRACTS OF MEDICINAL PLANT: MELIA
AZEDARACH L.
 ANTIBACTERIAL ACTIVITY OF ETHANOL EXTRACT OF MUSA PARADISIACA
CV. PUTTABALE AND MUSA ACUMINATE CV. GRAND NAINE; VENKATESH, KRISHNA
V*, GIRISH KUMAR K, PRADEEPA K, SANTOSH KUMAR S R.
 ANTIMICROBIAL ACTIVITY OF THREE DIFFERENT RHIZOMES OF CURCUMA
LONGA &CURCUMA AROMATICA ON UROPATHOGENS OF DIABETIC PATIENTS;
MEHVISH SALEEM*
1
, BETTY DANIEL
2
, MURLI K
3
 ANTIBACTERIAL ACTIVITY OF PLANT EXTRACTS AND
PHYTOCHEMICALS ON ANTIBIOTICRESISTANT BACTERIA; Gislene G. F.
Nascimento1*; Juliana Locatelli1; Paulo C. Freitas1,2; Giuliana L. Silva
 ANTIFUNGAL ACTIVITY OF MEDICINAL PLANT EXTRACTS AGAINST
PHYTOPATHOGENIC FUNGUS Alternaria SPP. Paola Díaz Dellavalle1 , Andrea
Cabrera1 , Diego Alem1 , Patricia Larrañaga1 , Fernando Ferreira2 , and Marco Dalla
Rizza1
 A comparative evaluation of antifungal activity of medicinal plant extracts
and chemical fungicides against four plant pathogens Harison Masih1 , JyotsnaKiran
Peter1* and Pratima Tripathi1

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