PRESENTATIO

N
ON
ANTIMICROBIAL SCREENING OF
BACTERIAL ENDOPHYTES ISOLATED
FROM Lantana camara
[Link](L.)

SUBMITTED BY- NIKITA PATHANIA(1641417)

NISHA CHOUDHARY(1741417)
SEMESTER- X
CENTAL UNIVERSITY OF JAMMU
SUBMITTED TO- [Link]
 TABLE OF CONTENTS
01 Introduction
 Plant endophytic 03 RESULT
associations
 Bacterial edophytes
 Equipments used in the
laboratory and
Materials
02 Methods
 Collection of the sample
04 CONCLUSION
 Surface sterilization and inoculation
 Subculture of endophytic bacteria
 Identification and characterization of bacteria
 Test microorganisms
 Evaluation of anti-microbial potential of
endophytes isolated from plant samples
 Screening of Endophytic bacteria for the detection
of anti-microbial potential

INTRODUCTION
In many developing countries like INDIA, about 80% of available therapeutic substances
are obtained from medicinal plants.

 The ethanomedicinal and scientific reports related with the medicinal properties
([Link], anti-rheumatic, biological control etc.) of Lantana camara designate it as
a valuable plant that establish itself as a candidate for the future drug development and
research purposes.

 An inventory of the endophytes of L. camara, its enzymes and metabolites is necessary

because they offer an alternative Sources of useful medicines for plant and animal
protection

 Endophytes are said to have an important role in regulation of plant growth and yield,
solubilizing essential elements and also suppressing pathogenic growth.

 According to studies endophytic microbes are present asymptomatically inside plant tissues
and may include bacteria, actinomycetes, fungi, protists and algae

 It has been elucidated through various experiments, the different endophytic associations
of Lantana with other plant microbes. Among these the common ones include bacterial and
fungal species.
Fig : General overview of the dynamics of plant-endophytic
 BACTERIAL ENDOPHYTES
● Bacterial colonizers living within the
plant tissues.

● Bacterial genera found as endophytes

are
Burkholderia,Methylobacterium,Sphigno
monas,Bacillus and many more
• They enter the plant tissues
through primary and lateral root
cracks , tissue wounds,lenticels.

• Helps in improving plant

growth,flower
scenescence,phytoremediation
EQUIPMENTS USED IN THE
LABORATORY
Weighing Balance: They are used in
precise weighing of small amounts (upto
miligrams) of samples and chemicals used
for preparing media and stock solutions.

Hot air oven : It is a device used in

sterilization. Oven uses dry heat to sterilize. It is
used to sterilize items that might be damaged
by moist heat (e.g., glassware like petriplates;
powders like starch; oils; metal eqipments like
scalpels.
Autoclave: An autoclave is a pressure chamber used to
sterilize equipment and supplies by subjecting them to
high pressure saturated steam at 121 °C for around
15–20 minutes depending on the size of the load and
the contents.
Used to sterilize culture media, discard, and other
equipments.

• Laminar flow Hood: It is an enclosed,

ventilated laboratory workspace for safely working
with materials contaminated with pathogens.

It is used in microbial inoculation and isolation studies

as well as sterile storage of materials. In addition, it is
utilized for protection of user, samples and the
environment from hazardous contamination and is
equipped with hepa filters and UV light.
Laboratory refrigerator: Is used for a wide variety of
purposes such as:
• maintenance and storage of stock culture between
subculturing periods.
• storage of sterile media to prevent dehydration.
• also used as repository for thermolable solutions, antibiotics
and serums.

BOD Incubator: It is a device used to grow

and maintain microbiological cultures.
• The incubator maintains optimal temperature,
humidity and other conditions such as the
carbon dioxide (CO2 ) and oxygen content of
the atmosphere inside.
Hot plate / stir plate: used to heat and stir
substances.
• It provides mixing and keeping the chemical
solutions and mixtures at a certain time and
temperature by the help of a magnetic bar.

Centrifuge Machine : is an apparatus that rotates at

high speed and separates substances of different
densities.
The centrifuge works using the sedimentation principle,
where the centripetal acceleration causes more dense
substances to separate out along the radial direction (the
bottom of the tube).
By the same principle, lighter objects will tend to move to the
top of the tube.
Microscope: It is an optical instrument consisting of a
combination of lenses which magnifies the image of the object seen
through it.
• It is used for the morphological study of a very small organisms
which are not visible by naked eye.
Micro= small scope=to view
Types of microscopes
 Simple
 Compound
 Electron

Micropipette: Piston driven air displacement pipettes are

micropipettes, which are tools to handle volumes of liquid in the micro
liter scale, and are the most accurate and precise pipettes. They are
more commonly used in biology and biochemistry.
The most common type of pipettes can be set to certain volume within its
operational range and are called adjustable. These pipettes commonly
have a label with their volume range like 10µl-100µl.
Inoculating loops and needles: Inoculating
loops are used to transfer microorganisms to growth media or for
staining slides.
• The wire forms a small loop with a diameter of about 5 mm.
• The loop of wire at the tip may be made of platinum or nichrome.
• Needles are straight wires (no loop) used to pick up
bacteria from closely packed colonies or to inoculate
in a very defined area.
needles commonly used to inoculate semi-soft media.

Glass slide: used to place specimens on to

observe under the microscope.
Cover slip: used to cover specimens on a
microscope slide.
Petri dishes: often used to make
agar plates for microbiology studies.
The dish is partially filled with media.

Test tubes: A test tube, also known as a culture

tube or sample tube, is a common piece
of laboratory glassware consisting of a finger-like
length of glass or clear plastic tubing, open at the top
and closed at the bottom used for making slants.
 MATERIAL AND METHODS
1. COLLECTION OF THE SAMPLE
Lantana camara [Link](L.)

Family : Verbenaceae

For the isolation of endophytic bacteria, healthy

leaves, stems, and roots of Lantana camara
[Link] L. were collected from wild and
cultivated plants at flowering stage from the
area of Hiranagar 5 Km from Jammu-Srinagar
highway with 32.486645 ° N and 75.268493°E
coordinates of district Kathua.
 SURFACE STERILIZATION AND INOCULATION
The root, stem and leaf sample of the Lantana plant were
taken and processed separately followed by washing
under running water to remove adhering soil particles.

For surface sterilization, samples were aseptically cut into

small sections and they were placed in 1% sodium
hypochlorite for 1 minute in petri plate.

•Repeated washing of plant samples at least 4 times in sterile distilled

water in the laminar air flow cabinet was done and dried on sterile filter
paper and one of the sterile distilled water sample is taken as control for
validation.

• Stem, root and leaf cuttings (9 each) were then

placed on nutrient agar medium.

• Plates with plant tissues were incubated at 28±2°C

for 24 hrs to recover the maximum possible colonies
of bacterial endophytes.
 SUBCULTURING
After 24 hrs. from the bacterial cultures, morphologically
different bacterial colonies were selected and are
repeatedly streaked on agar plates marked with
eight divisions to achieve bacterial isolates.

All selected isolates were subculture in nutrient agar

slants and finally, all the purified endophytes were
maintained at 4°C till further used. Pure cultures of
root, stem and leaf were obtained.
 IDENTIFICATION AND
CHARACTERIZATION OF
BACTERIA
 The morphology of a single cell colony is utilized to determine the
bacterial species.

 Bacteria were categorized as gram positive or negative using Gram

staining technique based on their shape and staining properties .

 Gram staining, which is frequently the first test conducted, uses

crystal violet or methylene blue as the predominant colour.

 Gram-positive organisms are those that preserve their primary

colour Part and appear purple brown under a microscope.

 Gram-negative organisms are those that do not take up primary

stain and look red under a microscope
 Fig: Gram staining of endophytic bacteria isolated from leaves of L. camara

All the bacteria of leaf sample were classified as gram positive and identification of
Bacillus bacteria due to formation of endospores.

Morphology- Arranged in chains, somewhere shrinked protoplast seen forming barrel

like structure, endospore formation.
 Fig: Gram staining of endophytic bacteria isolated from roots of L. camara

Bacteria were classified as Gram negative in SR1,3,4,5 and 6 with morphology including
long rods, arranged in chains.

Gram positive was identified in SR2 only with morphology including rod shaped.
 Fig: Gram staining of endophytic bacteria isolated from stem of L.
camara

Bacteria were classified as gram positive and identified as Bacillus due to the
formation of endospores.
 TEST MICROORGANISMS

The following human pathogenic bacteria were sub cultured on nutrient

agar media for further experimental usage:
Pseudomonas aeruginosa(MTCC96)
Salmonella typhimurium (MTCC 947)
Klebsiella pneumoniae(MTCC 109)

Bacterial endophytes were grown in 10ml slants containing 1ml

of nutrient broth(NB)medium each at 27±2° C for 24 hrs. After
24 hrs. culture medium with bacterial culture of each sample was
obtained and taken out in the Eppendorf and was centrifuged at
8000rpm for 10 minutes resulting in the separation of pellet and
supernatant. The supernatant was further stored and was used
for screening purpose.
 Evaluation of anti-microbial potential of
endophytes isolated from plant samples
• The antibacterial potential of endophytic bacteria was screened against three human
pathogenic bacteria with the help of agar well diffusion method.
• Under this method, a standardized concentration of inoculum with volume of 1ml is spread
evenly on the surface of gelled agar plates.
• A hole of 10mm in diameter is punched with a sterile cork borer aseptically under the laminar
air flow on the seeded plates.
• A fixed volume of endophytes from each sample(i.e. roots, stem and leaf) is then introduced
separately into the bored agar well and incubated at optimum temperature and for optimum
duration depending upon the test microorganism.
• Also,10ml of antibiotic(Streptomycin) was made and stored to be used as a negative control.
Screening of Endophytic bacteria for the
detection of anti-microbial potential
• The petri plates were then incubated in BOD at 37°C for 18hrs
and then the zone of inhibition was recorded in millimetres.

• Antimicrobial activity was assessed as growth inhibition of the

target organism around the agar wells through the appearance
of clear circular zone that was measured in millimetres.

• %inhibition was calculated through Kirby-Bauer method.

• (%) inhibition= (Rcontrol- Rtest)/Rcontrol x 100
• Rtest. = Colony diameter of pathogenic bacteria with antibiotic
isolate
• Rcontrol = Colony diameter of pathogenic bacteria

• 3 levels of susceptibility
A. Sensitive (S)- a normal dose of antibiotic is effective
B. Intermediate (I)- a higher than normal dose of an antibiotic is
required
C. Resistant (R)- no acceptable dose of antibiotic is effective.
 RESULTS

1. IN CASE OF STEM CULTURE

Fig: Petriplates with antimicrobial potential of endophytic bacteria
assessed against human pathogenic bacteria (i.e. Pseudomonas
aeruginosa (MTCC96) through appearance of zone of inhibition in
case of stem culture.
Fig: Petri plates with antimicrobial potential of endophytic bacteria
assessed against human pathogenic bacteria(i.e. Klebsiella
pneumoniae(MTCC 109)through appearance of zone of
inhibition in case of stem culture.
Fig: Petri plates with antimicrobial potential of endophytic bacteria assessed
against human pathogenic bacteria (i.e. Salmonella typhimurium (MTCC
947)through appearance of zone of inhibition in case of stem culture.
2. IN CASE OF LEAF
CULTURE
Fig: Petriplates with antimicrobial
potential of endophytic
bacteria assessed against
human pathogenic
bacteria(i.e. Pseudomonas
aeruginosa(MTCC96) through
appearance of zone of
inhibition in case of leaf
culture.
Fig: Petri plates with antimicrobial potential of endophytic bacteria assessed against human
pathogenic bacteria(i.e. Salmonellla typhimurium (MTCC 947)through appearance of
zone of inhibition in case of leaf culture.
Fig: Petriplates with antimicrobial potential of
endophytic bacteria assessed against human
pathogenic bacteria(i.e. Klebsiella
pneumoniae(MTCC 109)through appearance of
zone of inhibition in case of leaf culture.
3. IN CASE OF ROOT CULTURE
Fig: Petri plates with antimicrobial potential of
endophytic bacteria assessed against human
pathogenic bacteria(i.e. Salmonella
typhimurium (MTCC 947)through appearance of
zone of inhibition in case of root culture.
Fig: Petri plates with antimicrobial potential of
endophytic bacteria assessed against
human pathogenic bacteria (i.e. Klebsiella
pneumoniae(MTCC 109)through
appearance of zone of inhibition in case of
root culture
Fig: Petri plates with antimicrobial
potential of endophytic bacteria
assessed against human
pathogenic bacteria (i.e.
Pseudomonas
aeruginosa(MTCC96) through
appearance of zone of inhibition
in case of root culture.
 When tested against Streptomycin, maximum zone of inhibition was
produced by MTCC 947 i.e. Salmonella typhimurium followed by
MTCC96 i.e. Pseudomonas aeruginosa and then by MTCC 109 i.e.
Klebsiella pneumoniae.

 And among the samples of bacterial endophytes supernatants of

Lantana camara [Link] L. maximum zone of inhibition was
observed as 16mm, 13mm, 13mm in case of root, stem and leaf
respectively.

 Also maximum % of inhibition was shown against Salmonella

typhimurium (MTCC 947) in case of root sample i.e. 65.71%, in case
of stem sample 68.57% against Salmonella typhimurium (MTCC 947)
and in case of leaf sample 67.64% against Pseudomonas
aeruginosa(MTCC96).
 CONCLUSION
 From this study, it was found that the bacterial endophytes isolated from Lantana
camara [Link](L.) could suppress the growth of bacterial pathogens.

 The zone of inhibition is used to determine antibiotic susceptibility.

 Larger zones indicate reduced bacterial growth with greater antibiotic susceptibility,
while smaller zones indicate increased bacterial growth with lower antibiotic
susceptibility. If the antibiotic does not inhibit growth, there is no zone of inhibition and
therefore the bacteria are resistant.

 Following antimicrobial screening, the study on bioactive properties and compounds of

Lantana camara [Link](L.) associated with its bacterial endophytes can be
done. AndSuch compounds can be used as biocontrol agents against bacterial
phytopathogens.

 An inventory of the endophytes of L. camara, its enzymes and metabolites is necessary

because they offer an alternative sources of useful medicines for plant and animal
protection.
THANK YOU