Table of Contents :

1. Abstract

2. Historical Retrospect

3. Introduction

4. Materials and Methods

4. Result

5. Discussion

6. Conclusion

7. Acknowledgement

8. Reference
 ABSTRACT

This preliminary study aimed to investigate the antimicrobial role of the aqueous
extract of Tinospora cordifolia stem powder in a broth culture of Staphylococcus
aureus. The antimicrobial potential of natural products has garnered considerable
attention in the quest for alternative therapeutic agents against drug-resistant
pathogens. Tinospora cordifolia, a well-known medicinal plant in traditional
systems of medicine, has exhibited a wide array of therapeutic properties. In this
study, the inhibitory effects of the aqueous extract on the growth of
Staphylococcus aureus were evaluated. The results demonstrated significant
antimicrobial activity, as evidenced by the formation of a clear zone of inhibition
around the test tube containing the extract. The observed inhibitory effects
suggest that the bioactive compounds present in Tinospora cordifolia stem
powder have the ability to hinder the growth of Staphylococcus aureus. Further
research is warranted to identify and isolate the specific bioactive compounds
responsible for the observed antimicrobial activity and to determine the optimal
dosage and potential synergistic effects with existing antibiotics. Overall, this
preliminary study provides valuable insights into the antimicrobial potential of
the plant and highlights its potential as a natural alternative for the treatment of
Staphylococcus aureus infections.

Keywords : Tinospora cordifolia, antimicrobial, aqueous extract, stem powder,

Staphylococcus aureus
 HISTORICAL RETROSPECT
Throughout history, the search for effective antimicrobial agents to combat
infectious diseases has been a constant endeavor. Traditional systems of
medicine, rooted in ancient civilizations, have relied on natural remedies derived
from plants to treat various ailments. Tinospora cordifolia, a medicinal plant with
a rich historical background, has been recognized for its therapeutic properties in
traditional practices such as Ayurveda. The preliminary study on the
antimicrobial role of the aqueous extract of Tinospora stem powder against
Staphylococcus aureus builds upon this historical knowledge and aims to explore
its potential as a natural antimicrobial agent. This study represents a continuation
of the longstanding tradition of investigating the healing properties of plant-based
remedies. recent years, there has been a growing interest in validating the
traditional claims associated with Tinospora cordifolia and understanding its
therapeutic potential using modern scientific methods. The preliminary study on
its antimicrobial role against Staphylococcus aureus is part of this ongoing
exploration.
Sandhu et al. (2013) demonstrated the high efficiency of Tinospora cordifolia
against various microorganisms including Serratia marcescens, E. coli,
Salmonella typhi, Streptococcus thermophilus, Fusarium oxysporium, and
Aspergillus niger. However, it did not show any effect on Trichoderma reesei.
These findings suggest that Tinospora cordifolia has the potential to be a valuable
source of useful drugs. Further studies are necessary to isolate the active
components from the crude plant extract for proper drug development.
Molla et al. (2012) reported that TC-1, a chemical compound derived from
Tinospora cordifolia, exhibited a Minimum Inhibitory Concentration (MIC) of
128 g/ml against Bacillus megaterium and Salmonella typhi-A. The study also
indicated a strong hazardous impact of the chemical, as evidenced by a medium
lethal concentration (LC50) value of 9.34 g/ml.
In another investigation conducted by Nageswari et al. (2016), it was found that
T. cordifolia aqueous extracts displayed effective antimicrobial activity against
E. faecalis (28 mm) and S. typhi (26 mm) at a concentration of 50 mg/ml.
As research continues, further studies may delve deeper into Tinospora’s
historical context, exploring its traditional use in different cultures and its broader
therapeutic applications. This historical retrospect reminds us of the value of
ancient wisdom and the continuous journey towards unlocking nature's secrets
for the betterment of human health.
 INTRODUCTION

Tinospora cordifolia commonly known as Amrita (Guduchi) is a widely used plant in folk and
Ayurvedic systems of medicine. In Hindi, the plant is commonly known as ‘Giloya’ which is a
Hindu mythological term that refers to the heavenly elixir which has saved celestial beings
from old age and kept them eternally young. ‘Guduchi’, the Sanskrit name means one which
protects the entire body. The term ‘Amrita’ is attributed to its ability to impart youthfulness,
vitality and longevity (Bhandari, 2006).
Classification (Pullaiah, 1998)
The taxonomical hierarchy of T. cordifolia is as follows:
Kingdom : Plantae
Division : Angiosperma
Class : Dicotyledonae
Order : Ranunculales
Family : Menispermaceae
Genus : Tinospora
Species : T. cordifolia

STATE LANGUAGE VERNACULAR NAMES

Karnataka Kannada Amrita Balli

Tamil Nadu Tamil Shindilakodi
Andhra Pradesh Telugu Tippaatigo
Kerala Malayalam Ambrithu
Uttar Pradesh Hindi Giloy, Amrita
Maharashtra Marathi Gulavel
West Bengal Bengali Gulancha
Gujarat Gujarati Galo

Table 1 - Vernacular names of Tinospora cordifolia

A. Botanical description
Tinospora cordifolia is a large, deciduous, extensively spreading and climbing shrub
with several elongated twining branches. Stems are fleshy with thin, grayish or creamy
white colored bark and with long,
filiform and fleshy aerial roots from
the branches. Leaves are simple,
alternate, ovate or ovate chordate
which is 10-20 cm long and 8-15 cm
broad. The flowers are unisexual,
small on separate plants and
greenish yellow. In axillary and
terminal racemes or racemose
panicles, the male flowers are
clustered and female flowers are
usually solitary. The drupes are ovoid, glossy, succulent, red, pea sized and occur in
winter (Khosa and Prasad, 1971; Chopra, 1994). The seeds are curved. Fruits are
fleshy and single seeded. Flowers grow during the summer and fruits during the winter
(Kirtikar and Basu, 1975).
B. Origin and Distribution
Tinospora cordifolia is indigenous to the tropical areas of India, Myanmar and Sri Lanka
ascending to an altitude of 1200 m. It is a fairly common plant of deciduous and dry forests,
growing over hedges and small trees (Kirti et al., 2004; Bhandari, 2006).

C. Biophysical limits
They grow at low altitude up to 1030 m and preferably grow at sandy loam.
D. Uses of Tinospora cordifolia in traditional system of medicine Tinospora extracts are
widely used in the traditional system of medicine in the treatment of jaundice, rheumatism,
intermittent fevers, eye and liver ailmentanti-
spasmodic, anti-inflammatory, diabetes,
seminal weakness, urinary tract infections,
fever, general debility, skin diseases, and
expectorant, carminative, digestive, antistress
and aphrodisiac. It is also an important
constituent of many Ayurvedic formulations
and is reported to possess adaptogenic and
immunomodulatory activity in fighting
infections (Nayampalli et al., 1982). The stem
is bitter stomachic, diuretic (Nayampalli SS et
al., 1988), stimulates bile secretion, causes
constipation, allays (satisfies) thirst, burning
sensation, vomiting, enriches the blood and
cures jaundice. (Mishra p et al, 2014). The root and stem of T.cordifolia are prescribed in
combination with other drugs as an antidote in snake bite and scorpion sting (Nadkarni KM
et al., 1976; Zhao TF et al., 1991). Oral administration of an aqueous T. Cordifolia root extract
to alloxan diabetic rats caused a significant reduction in blood glucose and brain lipids
(Dhaliwal KS et al., 1999). Extract of T. cordifolia has also exhibited in vitro inactivating
 property against Hepatitis B and E surface antigen (Mehrotra R et al., 2000). In the
Antibacterial activity of aqueous, ethanol and chloroform extracts of leaves and stem of
Tinospora cordifolia (Hook.F.Thom) were tested on clinical isolates of urinary pathogens viz.,
Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa by disc
method. Ethanol extract of leaf showed greater inhibitory action than other tested extracts.
Indian system it is known to increase the longevity and the body’s resistance against various
diseases (Bhatt and Bhat, 1996).
D. Current Status
A number of chemical constituents belonging to different groups, viz., terpenoids like
tinosporide (Khuda et al., 1964), tinosporaside (Hanuman et al.,1986), furanolactone diterpene
(Khan et al., 1989), alkaloids like tinosporine (Khuda et al., 1964), berbrine (Pachey and
Schneidir, 1981) and steroids like β sitosterol
(Khaleque et al., 1970) have been isolated from T.
cordifolia stem and roots.
Some of the functional properties attributed to T.
cordifolia are as follows:
Anti-cancer activity - Administration of
intraperitoneal injection T. cordifolia stem
methanol extract to BALB/c mice (200 mg/kg
b.w.) for five days increased the total WBC count
significantly. It reduced solid tumor growth and
synergistically acted with cyclophosphamide in
reducing the animal tumors (Mathew and Kuttan,
1999).
Anti-diabetic and antihyperglycemic activity
Alcoholic extracts of the stem at a concentration of
50, 100 and 200 mg/kg b.w. caused a reduction in
the fasting blood sugar in the alloxan induced
Table 2: Plant Parts and Active diabetic rabbits (Wadood et al., 1992). Daily oral
Compounds feeding of T. cordifolia extracts for 40 days in
streptozotocin diabetic mice caused a reduction in
blood glucose concentration and also prevented polyurea (Grover et al., 2002).
Anti-inflammatory activity - The stem extract of T. cordifolia showed anti-inflammatory
activity on carrageenin induced hind paw edema in rats (Sharma and Singh, 1980). Antioxidant
activity Alcoholic extract of T. cordifolia roots administered at a dose of 100 mg/kg b.w. orally
to diabetic rats for six weeks normalized the antioxidant status of liver and kidney (Prince et
al., 2004).
Hypolipidaemic activity- Administration of aqueous extract of roots of T. cordifolia at a
concentration of 5 g/kg b.w. for six weeks resulted in a significant reduction in serum and tissue
cholesterol phospholipids and free fatty acids in alloxan diabetic rats (Stanely et al., 1999).
Immunomodulatory activity. The water and ethanol stem extracts of T. cordifolia inhibited
immunosuppression */produced by cyclophosphamide (Manjreker et al., 2000).
Hepatoprotective activity- The hepatoprotective activity of T. cordifolia extract has been
demonstrated in CCl4 induced liver damage in rats. The extract was found to normalize the
liver function as assessed by morphological, biochemical and functional tests (Bishayi et al.,
2002)
Anti-Oxidant Activity : Methanolic extract of stem of T. cordifolia has been reported to
anti-oxidant activity, by increasing the erythrocytes membrane lipid peroxide and catalase activity.
It also decreases the activity of SOD, GPx in alloxan induced diabetic rats. Extract of T. cordifolia
has been reported its free radical scavenging properties. Leaf extract of T. cordifolia reported to
have an alpha-glucosidase inhibitor, characterized as saponarin was found to be also significant
anti- oxidant and hydroxyl radical scavenging activity. Due to the presence of alkaloids it shows
protection against aflatoxin-induced nephrotoxicity [15]. T. cordifolia aqueous extract has a radio
protective activity, enhancing the survival of mice against a sub-lethal dose of gamma radiation.

Table 3: Major and sub groups of natural products present in different parts of Tinospora
cordifolia and their biological activities

Active Compound Plant Biological Activity (In Human

Component Part being)
Berberine, Choline, Anti-viral infections, Anti- cancer,
Tembetarine, Magnoflorine, anti-diabetes, inflammation,
Alkaloids Tinosporin, Palmetine, Stem, Neurological, immunomodulatory,
Isocolumbin, Aporphine Root psychiatric conditions
alkaloids, Jatrorrhizine,
Tetrahydropalmatine,

Furanolactone, Clerodane Vasorelaxant: relaxes norepinephrine

derivatives [(5R,10R)-4R- induced contractions, inhibits Ca++
8R- influx, anti-inflammatory, anti-
Diterpenoid dihydroxy-2S-3R:15,16- Whole microbial, anti- hypertensive, anti-
Lactones diepoxy-cleroda-13 (16), 14- Plant viral.
dieno-17,12S:18,1S- Induce apoptosis in leukemia by
dilactone], Tinosporon, activating caspase-3and bax, inhibits
Tinosporides, Jateorine, bcl-2.
Columbin

18-norclerodane
glucoside, Furanoid
diterpene glucoside, Treats neurological disorders like
Tinocordiside, ALS, Parkinsons, Dementia, motor
Tinocordifolioside, and cognitive deficits and neuron loss
Cordioside, in spine and hypothalamus,
Glycosides Cordifolioside Stem Immunomodulation, Inhibits NF-
Syringin, Syringin- kBand act as nitric oxide scavenger to
apiosylglycoside, Pregnane show anticancer activities.
glycoside, Palmatosides,
Cordifolioside A, B, C, D
and E

IgA neuropathy, glucocorticoid

induced osteoporosis in early
β–sitosterol, δ-sitosterol, 20 β- inflammatory arthritis, induce cell
Steroids hydroxyecdysone, Ecdysterone, Shoot cycle arrest in G2/M phase and
Makisterone A, Giloinsterol apoptosis through c-Myc suppression.
Inhibits TNF-
α, IL-1 β, IL-6 and COX-2.

Sesquiterpenoid Tinocordifolin Stem Antiseptic

Aliphatic Octacosanol, Whole Anti-nociceptive and anti-
 compound Heptacosanol Nonacosan-15-one plant inflammatory. Protection against 6-
dichloromethane hydroxydopamine induced
parkinsonisms in rats.
Down regulate VEGF and
inhibits TFN-α from binding to the
DNA.
3,(a,4-di hydroxy-3- methoxy-
benzyl)-4-(4- compounds
hydroxy-3- methoxy-benzyl)-
tetrahydrofuran, Jatrorrhizine, Root,
Others Tinosporidine, Cordifol, Whole Protease inhibitors for HIV and drug
Cordifelone, Giloinin, Giloin, N- Plant resistant HIV.
trans- feruloyltyramine as
diacetate, Tinosporic acid.

Table 4: Pharmaceutical products of T. cordifolia and their biological roles

Name of Market Product Biological Roles
Tinospora Cordifolia Pellets A number of diseases

Guduchi The immune system and the body's resistance to

infections
Abhaibhubejhr Anti-stress
Safe herb Cure by Anemia and sexual disabilities.
Brave Heart Capsule It lowers the lipid levels especially cholesterol and LDL-
cholesterol in body, diuretic
Cirrholiv capsules Hepatoprotective
Cirrholiv-ds syrup Hepatoprotective
Mussaffen Blood purifier and anti-allergic
MadhuMehari Cure by urinary problems, maintain blood sugar, fatigue
Tonplex Increase immunity
Rebuild Anti- stress and anti- oxidant

Keeping in view the above-mentioned medicinal properties, Tinospora cordifolia has been listed
an important plant amongst the topmost medicinal plants by NMPB, New Delhi, Government of
India. Various funding agencies have been sanctioned the large amount to research on this
important ignored plant species in the form of major research projects to different organizations/
institutions, are also being represented here for the help of scientists.

At the same time, type of research work done on this potent medicinal plant species are also being
given here
Tinospora cordifolia was selected for our study due to its various functional
properties as listed above and also due to its wide usage in Ayurvedic formulations.

➢ In vivo and in vitro research investigations on T. cordifolia have shown the use of its stem and
roots and not the leaves. Not many studies have been carried out to show the antimicrobial,
 antioxidant and antidiabetic activities of its leaves. Antifungal activity studies against
dermatophytes have not been proved till now even though its activity against the field fungi
have been carried out. Moreover, in this study we are trying to select a suitable solvent extract
of Stem which has antimicrobial, antioxidant and antidiabetic activity.
➢ The scientific research on Tinospora cordifolia suggests a huge biological potential of
this plant. T. cordifolia is widely used in ayurvedic medicine for the treatment of various
ailments. It is reported that extract of T.cordifolia has good immunomodulating effect. It
also has the ability to scavenge free radicals induced membrane damage. It also has the
hypoglycemic activity and hypolipidemic activity. It also has ability to protect the liver
from various diseases. It is found that it is non-toxic in acute toxicity studies. Various types
of studies, which have been done on T. cordifolia, reveal that it is an excellent drug, which
could be a good remedy for various aliments of animals as well as human beings yet the
safety and the potential indications in human beings and animals have to be established
using modern techniques.
All these facts have prompted us to select only the stem extract of T. cordifolia for our study.

Increasing Bacterial Infections with respect to Staphylococcus aureus

In recent years, the world has witnessed a concerning rise in bacterial infections, posing significant
challenges to public health. Several factors contribute to this increase, and it is important to address
them to mitigate the impact on individuals and communities. The global increase in bacterial infections
has also had an impact on the prevalence of Staphylococcus aureus (S. aureus) infections.

STAPHYLOCOCCUS AUREUS
In 1881, Sir Alexander Ongston, a Scottish surgeon,
discovered that Staphylococcus can cause wound
infections after noticing groups of bacteria in pus from a
surgical abscess during a procedure he was performing.
He named it Staphylococcus after its clustered
appearance evident under a microscope. Then, in 1884,
German scientist Friedrich Julius
Rosenbach identified Staphylococcus aureus,
discriminating and separating it from Staphylococcus
albus, a related bacterium. In the early 1930s, doctors
began to use a more streamlined test to detect the presence Domain: Bacteria
of an S. aureus infection by the means
of coagulase testing, which enables detection of an
Phylum: Bacillota
enzyme produced by the bacterium. Prior to the 1940s, S.
aureus infections were fatal in the majority of patients.
However, doctors discovered that the use of penicillin Class: Bacilli
could cure S. aureus infections. Unfortunately, by the end
of the 1940s, penicillin resistance became widespread
amongst this bacterium population and outbreaks of the Order: Bacillales
resistant strain began to occur.

Family: Staphylococcaceae

Genus: Staphylococcus
STRUTCURE
Staphylococci are Gram-positive cocci about 0.5 – 1.0 μm in diameter. They grow in clusters, pairs and
occasionally in short chains. The clusters arise (tend to be arranged in clusters that are described as
“grape-like.”) because staphylococci divide in
two planes. The configuration of the cocci
helps to distinguish micrococci and
staphylococci from streptococci, which usually
grow in chains. Observations must be made
on cultures grown in broth, because
streptococci grown on solid medium may
appear as clumps. On media, these organisms
can grow in up to 10% salt, and colonies are
often golden or yellow (aureus means golden or
yellow). These organisms can grow aerobically
or anaerobically (facultative) and at
temperatures between 18 C and 40 C. Typical
biochemical identification tests include catalase
positive (all pathogenic Staphylococcus species),
coagulase positive (to
distinguish Staphylococcus aureus from
other Staphylococcus species), novobiocin
sensitive (to distinguish
from Staphylococcus saprophyticus), and
mannitol fermentation positive (to distinguish
from Staphylococcus epidermidis).
EPIDEMIOLOGY
S aureus is notorious for causing boils, furuncles,
styes, impetigo and other superficial skin
infections in humans. It may also cause more
serious infections, particularly in persons
debilitated by chronic illness, traumatic injury,
burns or immunosuppression. These infections
include pneumonia, deep abscesses, osteomyelitis,
endocarditis, phlebitis, mastitis and meningitis,
and are often associated with hospitalized patients
rather than healthy individuals in the
community. S aureus and S epidermidis are common causes of infections associated with indwelling
devices such as joint prostheses, cardiovascular devices and artificial heart valves. Staphylococcus
aureus (including drug-resistant strains such as MRSA) are found on the skin and mucous membranes,
and humans are the major reservoir for these organisms. It is estimated that up to half of all adults are
colonized, and approximately 15% of the population persistently carry S. aureus in the anterior nares.
Some populations tend to have higher rates of S. aureus colonization (up to 80%), such as health care
workers, persons who use needles on a regular basis (i.e., diabetics and intravenous (IV) drug users),
hospitalized patients, and immunocompromised individuals. S. aureus can be transmitted person-to-
person by direct contact or by fomites S. aureus is both a frequent commensal and a leading cause
of endocarditis, bacteraemia, osteomyelitis and skin and soft tissue infections. With the rise of hospital-
based medicine, S. aureus quickly became a leading cause of health-care-associated infections as well.
Penicillin offered short-lived relief: resistance arose in the 1940s, mediated by the β-lactamase
gene blaZ. The first semi-synthetic anti-staphylococcal penicillins were developed around 1960
and methicillin-resistant S. aureus (MRSA) was observed within 1 year of their first clinical use. In fact,
genomic evidence suggests that methicillin resistance even preceded the first clinical use of anti-
staphylococcal penicillins2. Methicillin resistance is mediated by mecA and acquired by horizontal
transfer of a mobile genetic element designated staphylococcal cassette chromosome mec (SCCmec)3.
The gene mecA encodes penicillin-binding protein 2a (PBP2a), an enzyme responsible for crosslinking
the peptidoglycans in the bacterial cell wall. PBP2a has a low affinity for β-lactams, resulting in
resistance to this entire class of antibiotics. The first-line of natural defense against S. aureus is the
native immune system and the next line of defense against S. aureus infection is the use of antibiotics,
but as with all antibiotics misuse or overuse of these agents has in part lead to resistant strains of S.
aureus. In order to effectively treat S. aureus, new strategies and technologies need to be developed.
Unfortunately, little work has been done since the 1970s to investigate and develop new classes of
antibiotics by the pharmaceutical industry (Wenzel, 2004). Since 2000, only two new classes of
antibiotics have been approved for the treatment of gram-positive bacteria: the oxazolidinones
(linezolid) and the cyclic lipopeptides (daptomycin) (Wenzel, 2004) but these drugs have yet to be
widely used by the medical industry. Moreover, reports of resistance to these antibiotics among S.
aureus isolates emerge within a year of their approval for use in humans (Mangili & Segreti, 2005)

The Project Work Aims at –

1. Providing a comprehensive overview of the research conducted on the antibacterial
properties of Tinospora cordifolia
2. Presenting the experimental methodology employed to evaluate the antibacterial effects,
including the extraction process and growth conditions
3. Demonstrating the results obtained from the study, highlighting the observed antibacterial
effects of Tinopora cordifolia on Staphylococcus aureus.
4. Discussing the significance of the findings and their potential implications for the
development of alternative antibacterial agents.
5. Analyzing the potential mechanisms of action underlying the observed antibacterial effects,
drawing upon existing research and theories.
6. Addressing any limitations or challenges encountered during the study and suggest possible
avenues for future research.
7. Engaging the audience through effective visual aids, clear explanations, and concise delivery
to ensure a comprehensive understanding of the study's outcomes.
By accomplishing these objectives, this pilot study aims to contribute to the existing body of
knowledge on natural antibacterial agents and encourage further exploration of Tinospora
cordifolia's potential applications in combating Staphylococcus aureus infection.
 Materials Required For The Study

CHEMICALS Nutrient Agar (HIMEDIA)

Tinospora cordifolia Stem
powder or Organic Giloy
Powder (CARMEL Organics)

OTHER Distilled water

MATERIALS PBS (Phosphate buffer saline)
Ethanol (C2H5OH) (70%)
Spirit

BACTERIA Agar Plate culture of

AND Staphylococcus aureus was a
kind gift from Dr. M. Mandal
CULTURE Madam, Prof. Microbiology
CONDITIONS Department, Dinabandhu
Andrews College.
It was stored at 37°C in B.O.D
incubator in our laboratory.

Commercial Purchased Organic Giloy

Powder, Nutrient Broth and Agar Plate
Culture Of Staphylococcus Aureus

INSTRUMENTS:

Glass goods Plastic Straw Micropipette

(25µml)
Tip box Paper disc Spirit Lamp and
lighter
Cotton Plastic tray Brown paper
Rubber bands Marker pen Scissor
Forceps Mask Conical flask
Glass beaker Aluminium foil Spatula
Whatman no. 1 filter paper Funnel
Petri plate set (glass)(10.16cm) Lshaped glassrod (spreader)
Inoculation loop Conical flask Glass beaker Conical Flask, Measuring Cylinder
and Figure showing components of
Measuring Cylinder (100ml and 50ml) Aluminium foil Nutrient Broth(500g)
 The following procedure has been carried out in order to observe the Antimicrobial
effects of T.cordifolia stem powder in Broth Culture of Staphylococcus Aureus :
Preparation of Tinospora cordifolia stem extract:
Commercially purchased Giloy Stem Powder (Tinospora cordifolia), manufactured and marketed by
Carmel Organics Private Limited, was taken. A total of 4 grams of the stem powder was soaked with
50 mL of distilled water in a sterilized glass jar and stirred to ensure proper mixing. The mixture was
then stored at a temperature of 4°C for a period of 15 days. Prior to 15 days, the mixture was subjected
to filtration using Whatman No. 1 filter paper to obtain the extract. The resulting extract was then
transferred to a sterilized conical flask, which was sealed with aluminum foil, and stored at 4°C until
further use.

FIGURE – Extraction Preparation of Tinospora

cordifolia stem powder

Preparation of Culture Media:

Microbial culture media preparation is the process of mixing nutrients, agents for buffering and
maintaining the osmotic balance, as well as selective inhibitors or indicators to create an agar or broth
that supports the growth and the differentiation of microorganisms. For the following experiment,
Nutrient Broth has been utilized. Nutrient broth is a general purpose medium used for the cultivation of
a wide variety of fastidious and non-fastidious microorganisms with non-existent nutritional
requirements. Peptone and yeast extract provide nitrogen compounds, vitamin B complex, amino acids
and other nutrients essential for growth. The relatively simple formulation of nutrient broth is ideal for
the cultivation of microorganisms that are not specific in their nutritional requirements. The medium
consists of beef extract (or yeast extract), peptone and sodium chloride. To facilitate media
preparation, a carefully measured amount of broth powder (3.1 g) was combined with 100 ml of
distilled water in a conical flask. The mixture was gently stirred and securely covered with
aluminium foil. The media formulation process involved preparing the media in separate conical
flasks. These flasks were then sealed with a cotton plug and securely wrapped with glossy brown
paper, held in place with a rubber band. Next, the media-containing flasks underwent autoclave
sterilization at a pressure of 15 pounds for 15 minutes at a temperature of 121 degrees Celsius.
After the completion of the autoclaving process, the conical flask was carefully retrieved from the
autoclave and allowed to cool down to a temperature range of approximately 40-45°C. Following the
sterilization procedure, the media was safely transferred into previously sterilized test tubes.
 For further experimental procedure, the following machineries present in the laboratory
have been imperative:

Autoclave Sterilization of Glassware and Prepared Media in the study:

When microbiological media has been made, it still has to be sterilized because of microbial
contamination from air, glassware, hands, etc. Within a few hours there will be thousands of bacteria

Figure Showing Sterilization of Glassware and Prepared

Media (Left) And Stem Powder Extract of Tinospora
cordifolia post Autoclave (Right)

reproducing in the media so it has to be sterilized quickly before the microbes start using the nutrients
up. The sterilization process is a 100% kill, and guarantees that the medium will stay sterile unless
exposed to contaminants by less than adequate aseptic technique to exposure to air. Media sterilization
is carried out with the autoclave, basically a huge steam cooker. Autoclaving is a method of sterilizing
with water vapor under pressure. Cotton plugs, gauze, labware, plastic caps, glassware, filters, pipettes,
water, and nutrient media can all be sterilized by autoclaving. Nearly all microbes are killed by exposure
to the super-heated steam of an autoclave for 10-15 minutes. Steam enters into a jacket surrounding the
chamber. When the pressure from the steam is at a certain point in the jacket, a valve allows the steam
to enter the chamber. The pressure will go up over 15 pounds per square inch (psi): at this point the
timer begins to count down--- usually for 15 minutes, depending on the type of media. The high pressure
in a closed container allows the temperature to go above the highest temperature one could get by just
boiling, around 121⁰C. Therefore, the parameters for sterilization with an autoclave are 121⁰C at >15
psi for 15 minutes. Fifteen minutes is the thermal death time for most organisms (except some really
hardy spore formers).The prepared media is distributed in different ways, depending on the form one is
making. Broths and agar deeps are dispensed into tubes and then sterilized. In this study, an
autoclave sterilizer was utilized to ensure the sterility of the required glassware and prepared media.
For the sterilization process, the prepared media, L-shaped glass rod, Petri plates (washed properly
with cleansing agent and rinsed several times under tap water) conical flasks, and tip box were
carefully packed using cotton, brown paper, and secured with rubber bands. Prior to the sterilization
process, it was ensured that the internal chamber
of the autoclave was clean, and the water level
was maintained above the designated stand inside
the autoclave. The packed instruments were then
placed on a plate that had been previously
positioned within the autoclave. Next, the lid of
the autoclave was firmly closed and secured with
its screw knobs, while the pressure release valve
was tightened as well. The autoclave was then
powered on, and the pressure gauge located on
the top of the lid was monitored. The normal
duration for the autoclave to reach a pressure of
Autoclaved Glassware and Prepared 15 psi (pounds per square inch), which
Media corresponds to a temperature of approximately
121°C inside the chamber is approximately 30-40
minutes. Once the desired pressure was achieved, the autoclave was maintained in this state for 15
minutes to ensure effective sterilization, after which it was switched off. The release valve was then
loosened, allowing the pressure to escape safely. After the completion of the autoclaving process,
the conical flask was carefully retrieved from the autoclave and allowed to cool down to a
temperature range of approximately 40-45°C. Following the sterilization procedure, the media
was safely transferred into previously sterilized test tubes. Simultaneously, Forceps were also
sterilized by dipping in 70% ethanol.

Sterilization via Laminar Air Flow System:

A laminar flow cabinet or tissue culture

hood is a carefully enclosed bench designed to
prevent contamination of semiconductor wafers,
biological samples, or any particle sensitive
materials. A Laminar Air Flow hood consists of
a filter pad, a fan, a HEPA (High Efficiency
Particulates Air) filter and UV light. Air is
drawn through a HEPA (high-efficiency particulate
air) filter and blown in a very smooth, laminar
flow towards the user. Due to the direction of air
flow, the sample is protected from the user but
the user is not protected from the sample. The
cabinet is usually made of stainless steel with
no gaps or joints where spores might collect.
The fan sucks the air through the filter pad where dust is LAMINAR FLOW SYSTEM
trapped. After that the prefiltered air has to pass the HEPA
filter where contaminating fungi, bacteria, dust etc. are removed. Sterile air flows into the
working area where work can be done without contamination. The constant flow of filtered air
provides material and product protection. For the study, prior to commencing the task, the
laminar air flow system was activated and allowed to operate for a duration of 30 minutes. The
laminar air flow unit underwent a thorough cleaning procedure using 70% alcohol.
Additionally, the UV light within the unit was switched on for a period of 20 minutes. The fan
of the laminar air flow system remained operational for 15 minutes.

Aforesaid procedures are then followed by Culture Preparation and

Inoculation of Bacteria via Broth Dilution Assay:

Broth dilution is a technique in which

containers holding identical volumes of broth
with antimicrobial solution in incrementally
(usually geometrically) increasing
concentrations are inoculated with a known
number of bacteria. The aim of broth dilution
method is to determine the lowest
concentration of the assayed antimicrobial
agent (minimal inhibitory concentration,
MIC) that, under defined test conditions,
inhibits the visible growth of the bacterium
being investigated.

Culture Media of Staphylococcus

aureus

The word inoculation comes

from the Latin
word ‘inoculare’ which has the
meaning ‘to graft’. Inoculation
is the purposeful introduction of
bacteria into a sterile growth
medium. Inoculation of bacteria
is achieved through sterilized L-
Inoculation loop
shaped glass rod/ spreader or by
using inoculation loop. Initially, the loop was subjected to the spirit lamp until
the wire reached a red-hot temperature. Subsequently, the loop was carefully
touched to the edge of the agar plate to facilitate cooling. A small quantity of
bacteria was gently collected by scraping. Next, the loop was used to streak the
surface of the agar media, and the plate was promptly covered with its lid. To
prevent contamination, the loop was once again heated until it achieved a red-hot
state.
Evaluation of Antibacterial Activity was conducted using the Broth Dilution
Method under aseptic conditions in a laminar airflow environment. The
experimental setup included four sterilized test tubes with cotton plugs: two
control tubes labelled as C1 and C2, and two tubes designated as Ts1 (containing
Media+ Extract dose 600µl+Inoculums) and Ts2 (containing Media+ Extract
dose 1000 µl). Each of the four test tubes was filled with an equal volume of
nutrient broth media. Micropipettes were used to add the plant stem extract
(T.cordifolia) at doses of 600 µl and 1000 µl to Ts1 and Ts2 respectively,
followed by thorough mixing. The inoculating loop then was heated until it turned
red and then allowed to cool. One loopful of the test bacteria, S. aureus, was
carefully transferred to each of the four test tubes. The contents were briefly
mixed, and the loop was removed without touching the rim of the tube. To ensure
airtight conditions, the test tubes were tightly closed with cotton plugs and placed
in a sterilized beaker for 24 hours. Afterward, the loop was flamed again and
allowed to cool down. The results were observed and recorded after 24 and 48
Hours of incubation, and photographs were taken.

RESULTS AND OBSERVATION:

➢ Inhibitory Effects of Aqueous extract of Tinospora cordifolia stem powder at a
dose of 600 µl

FIGURE- 24 HOURS OBSERVATION FIGURE 48 HOURS OBSERVATION

(Control and Experimental Tube) (Control and Experimental Tube)
 CONTROL (C1) Experimental tube (Ts1)

Appears slightly cloudy post (Fig.1 Post 1. Growth of a ring or pellicle can be seen clearly.
24Hours)
2. The growth of bacteria appeared to be relatively
lower compared to the control.
(Fig.1 Post 24Hours)

1. Consistent cloudiness (visually more No significant result was observed

significant)
Growth rate seemed to be lower
2. Formation of a layer of deposited
than the standard control and no death cell was
pellicle on the surface
observed.
3. Observation of deceased cells
(Fig.2 Post 48Hours)
(Fig.2 Post 48Hours)

Table : Inhibitory Effects of Aqueous extract of Tinospora cordifolia

stem powder at a dose of 600µl

The antibacterial effect of Tinospora cordifolia was assessed by visually comparing the results after 24
and 48 hours of exposure. The stem extract of Tinospora cordifolia was used at a concentration of
600μL in water. The observations revealed that the extract exhibited a significant antimicrobial activity
against Staphylococcus aureus. In the control test tube, a cloudy appearance was observed, indicating
the presence of bacterial growth. This cloudiness can be attributed to the proliferation of Staphylococcus
aureus in the absence of any inhibitory substance. The cloudy appearance serves as a visual indication
of bacterial growth and the absence of any inhibitory effect on the pathogen. On the other hand, the
experimental test tube containing the stem extract of Tinospora cordifolia exhibited a glossy
appearance. This glossy appearance suggests that the growth of Staphylococcus aureus was inhibited in
the presence of the extract. The absence of visible particles or precipitates in the extract indicates that
it is free from insoluble compounds or suspended solids. This glossy appearance further supports the
antimicrobial activity of the Tinospora cordifolia extract, indicating its ability to suppress the growth
of the bacteria.Overall, the visual comparison of the control and experimental test tubes clearly
demonstrates the effectiveness of the 600 μL concentration of Tinospora cordifolia stem extract against
Staphylococcus aureus. The cloudy appearance in the control tube signifies the bacterial growth, while
the glossy appearance in the experimental tube suggests the inhibitory effect of the extract on the
bacteria. These findings provide valuable insights into the antimicrobial potential of Tinospora
cordifolia as a natural alternative for combating Staphylococcus aureus infections.
 ➢ Inhibitory Effects of Aqueous extract of Tinospora cordifolia stem powder at a
dose of 1000 µl

FIGURE- 24 HOURS OBSERVATION FIGURE 48 HOURS OBSERVATION

(Control and Experimental Tube) (Control and Experimental Tube)

The results indicated that the higher concentration of 1000μL showed more significant
inhibitory effects on bacterial growth compared to the lower concentration of 600μL. This
observation suggests that increasing the concentration of the Tinospora cordifolia extract led
to enhanced antimicrobial activity against the bacteria, possibly due to a higher concentration
of bioactive compounds present in the extract. Thus, an important aspect of the experiment was
the visual appearance of the test tubes containing the different concentrations of the extract.
The test tube with the higher concentration (1000μL) displayed a more glossy appearance
compared to the previous test tube, indicating a clear and transparent solution without any
visible particles or precipitates. This glossy appearance suggests that the higher concentration
extract inhibited the growth of the bacteria more effectively. On the other hand, the control test
tube, which did not contain the Tinospora cordifolia extract, exhibited a cloudy appearance.
This cloudiness suggests that the bacteria in the control group continued to grow and proliferate
without inhibition. The lack of a glossy appearance in the control test tube further reinforces
the importance of the Tinospora cordifolia extract in inhibiting bacterial growth. These
observations provide valuable insights into the antimicrobial properties of the Tinospora
cordifolia extract. The increase in concentration resulted in a more pronounced inhibitory effect
on bacterial growth, as evidenced by the glossy appearance of the test tube. The cloudy
appearance of the control test tube indicates the ongoing growth of bacteria in the absence of
the extract. Overall, these findings highlight the potential of Tinospora cordifolia as a natural
antimicrobial agent and emphasize the importance of concentration in determining
its effectiveness against bacteria like Staphylococcus aureus.

CONTROL (C1) Experimental tube (Ts1)

Appears slightly cloudy 1. Growth of a ring or pellicle can be seen clearly.

(Fig.1 Post 24Hours) 2. The growth of bacteria appeared to be
relatively lower compared to the control.
(Fig.1 Post 24Hours)

1. Consistent cloudiness (visually more No significant result was observed

significant)
Growth rate seemed to be lower
2. Formation of a layer of deposited
than the standard control and no death cell was
pellicle on the surface
observed.
3. Observation of deceased cells
(Fig.2 Post 48Hours)
(Fig.2 Post 48Hours)

Table : Inhibitory Effects of Aqueous extract of Tinospora cordifolia stem

powder at a dose of 1000µl

DISCUSSION
The current study aimed to investigate the antimicrobial effects of Tinospora cordifolia stem
powder in a broth culture of Staphylococcus aureus. By observing the growth inhibition and
other relevant parameters, valuable insights were gained regarding the potential of Tinospora
cordifolia as an antimicrobial agent against this particular bacterium. The results of the study
demonstrated that the stem powder of Tinospora cordifolia exhibited significant antimicrobial
effects against Staphylococcus aureus in the broth culture. The observed inhibition of bacterial
growth indicates that the bioactive compounds present in the stem powder have the ability to
hinder the proliferation of Staphylococcus aureus. The antimicrobial effects of Tinospora
cordifolia can be attributed to the presence of secondary metabolites and phytochemicals in the
plant material. These include quinones, polyphenols, alkaloids, flavonoids, tannins, coumarins,
terpenoids, lectin, and polypeptides. Each of these compounds may contribute to the
antimicrobial action through different mechanisms. For example, quinones and flavonoids have
been shown to bind to adhesins, form complexes with the bacterial cell wall, and inactivate
bacterial enzymes. Terpenoids, polyphenols, and tannins, on the other hand, can disrupt
bacterial membranes and form metal ion complexes that render the bacteria inactive. The
observed antimicrobial effects of Tinospora cordifolia stem powder also indicate its potential
as a natural alternative to conventional antibiotics.
 CONCLUSION
With the growing concern of antibiotic resistance, exploring the antimicrobial
activity of plant-based substances like Tinospora cordifolia becomes even more
crucial. Harnessing the therapeutic potential of natural compounds may offer new
avenues for the development of effective antimicrobial agents. As researchers
delve deeper into the mechanisms of action and conduct clinical trials, giloy plant
may emerge as a valuable addition to the repertoire of natural remedies,
contributing to improved health and well-being. Furthermore, the observed
antimicrobial effects of T. cordifolia stem powder provide a foundation for
further investigations. Future studies could focus on identifying and isolating the
specific bioactive compounds responsible for the observed antimicrobial activity.
Determining the minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) of the stem powder could help establish the
optimal dosage required for effective inhibition of Staphylococcus aureus growth.
Additionally, evaluating the potential synergistic effects of Tinospora with
existing antibiotics could provide insights into combination therapies that may
enhance antimicrobial efficacy. Further research is necessary to fully elucidate
the underlying mechanisms and optimize its application as a natural alternative
to combat bacterial infections.

In conclusion, this preliminary study has shed light on the antimicrobial potential
of the aqueous extract of Tinospora Cordifolia stem powder against
Staphylococcus aureus in a broth culture. The results have demonstrated
significant inhibitory effects on bacterial growth, indicating the promising role of
Tinospora Cordifolia as a natural antimicrobial agent. The dose-dependent
response observed, with the higher concentration (1000 μL) of the extract
showing enhanced inhibitory effects compared to the lower concentration (600
μL), suggests that the effectiveness of the extract is influenced by its
concentration. Further studies could explore the optimal concentration and
determine the minimum inhibitory concentration required for effective
antimicrobial activity. The glossy appearance of the test tube containing the
higher concentration of the extract indicates a greater inhibition of bacterial
growth, while the cloudy appearance of the control test tube highlights the
ongoing proliferation of bacteria in the absence of inhibitory substances. These
visual observations further support the antimicrobial activity of the Tinospora
Cordifolia extract.

Further research and development are warranted to fully harness the therapeutic
potential of this natural resource and translate it into practical applications for the
treatment of infectious diseases.
 ACKNOWLEDGEMENT

I am immensely grateful to Dr. Maitrayee Mondal, Assistant Professor in the Department of

Microbiology, Dinabandhu Andrews College, for generously providing the bacterial culture
used in this study. Her kind gesture and contribution have been invaluable to the success of this
research. I extend my heartfelt appreciation to all those who played a pivotal role in this project.
My deepest gratitude goes to our respected Professors, especially Dr. Aryya Mitra and Dr.
Antara Banerjee. I further extend gratitude to Dr. Sangeeta Sengupta, and Prof. Poulami
Mukherjee, Department of Zoology, Bangabasi College, for their unwavering guidance and
support throughout the completion of this project. Their constant inspiration, timely
suggestions, and encouragement have been instrumental in its accomplishment. I would like to
extend my heartfelt acknowledgments to senior and respected teachers, including Dr. Sanjib
Ghoshal, Dr. Rupa Mukhopadhyay, Dr. Kabita Kundu, Dr. Sudipta Mandal, Dr. Suman
Bhattacharyya, Mr. Bibhas Kora, and Mrs. Mamer Pal, Professors at Bangabasi College, for
their valuable support and guidance. Furthermore, I am grateful to our esteemed Principal, Dr.
Himadri Bhattacharyya Chakrabarty, for providing an environment conducive to learning
and research. I would like to extend my sincere appreciation and heartfelt thanks to all the
esteemed faculty members and non-teaching staff of our department for their unwavering
support and exceptional cooperation throughout this project. Their consistent guidance,
valuable insights, and cooperation have been instrumental in the successful completion of this
study. I am truly grateful for their contributions and dedication to fostering a conducive
research environment.

To everyone who contributed to this project and stood by me with unwavering support, thank
you so much. Your assistance has been indispensable, and I am deeply grateful for all your
efforts.
 REFERENCES

1. Deinhardt-Emmer S et al. 2018. Virulence patterns of Staphylococcus aureus strains from

nasopharyngeal colonization. J Hosp Infect 100:309–315.
2. Lowy FD. 1998. Staphylococcus aureus infections. N Engl J Med;339(8):520-32. [PubMed])
3. Krismer B et al. 2017. The commensal lifestyle of Staphylococcus aureus and its interactions
with the nasal microbiota. Nat Rev Microbiol 15:675–687.
4. Tong SYC et al. 2015. Staphylococcus aureus infections: epidemiology, pathophysiology,
clinical manifestations, and management. Clin Microbiol Rev 28:603–661.
5. Reagan DR et al. 1991. Elimination of coincident Staphylococcus aureus nasal and hand
carriage with intranasal application of Mupirocin Calcium ointment. Annals of Internal Medicine
114:101–106.
6. Leonti M et al. 2014. food of the immortals according to the Bower Manuscript (Kashmir, 6 th
century AD). J Ethnopharmacol ; 155(1): 373-86.
7. Mishra R, Kaur G. 2013. Aqueous ethanolic extract of Tinospora cordifolia as a potential
candidate for differentiation based therapy of glioblastomas. PLoS One ; 8(10): e78764.
8. Dhama K et al. 2013 Global warming and emerging infectious diseases of animals and
humans: Current scenario, challenges, solutions and future perspectives- A review. Int J Curr
Res: 5(7): 1942-58.
9. MM. Pandey, S. Rastogi, AK. Rawat, “ Indian herbal drug for general healthcare: An
overview”, Internet Journal of Alternative Medicine, 2008.
10. Singh SS et al. 2003. Chemistry and medicinal properties of Tinospora cordifolia (Guduchi).
U J. Pharmacol. 35:83-91.
11. Singh R et al. 2016 De novo transcriptome sequencing facilitates genomic resource
generation in Tinospora cordifolia. Funct Integr Genomic ; 16(5): 581-91.
12. Saha S and Ghosh S. 2012. Tinospora cordifolia: One plant, many roles. AncSci Life, 31(4):
151-59.)
13. Mathew S, Kuttan G (1999). Immunomodulatory and antitumour activities of Tinospora
cordifolia. Fitoterapia. 70(1):35-43.)
14. Molla AH, Zakaria MG, Molla MT, et al. Chemical investigation and Microbial activity of
Tinospora cordifolia miers. Journal of bio-science. 2012; 20:153-60.
15. Nageswari G et al. 2016. Antibacterial activity of Tinospora cordifolia extracts on clinical
isolates from HIV infected patients. Int J Curr Res.8(6):33072-7.
16. Sandhu A et al. 2013 Antimicrobial Activity and Photochemical Screening of Tinospora
cordifolia and Euphorbia Hirta.