3

DECLARATION
I hereby, Avantika Sharma declare that I have done my project on the topic “Cultivation of bacteria on
newly formulated plant based media” which is submitted in partial fulfilment of the requirement for the
degree of Masters of Science in Microbiology, Department of Microbiology, Himachal Pradesh
University, Summerhill Shimla the data included in it is pure and is a record of our original piece of
research work and it is worthy of the consideration for the degree of M.Sc. Microbiology. The assistance
and help that has been received during the course of this investigation has been duly acknowledged.
Date:
Place:
Avantika Sharma
 5

CONTENTS

Chapter Title Pages

1. Introduction

2. Review Of Literature

3. Materials And Methods

4. Results And Discussion

5. Summary

6. References

Annexures
 6

ABBREVATIONS
E. coli Escherichia coli
S. aureus Staphylococcus aureus
C. albicans Candida albicans
C. dubliniensis Candida dubliniensis
EMB Eosin Methylene Blue
MSA Mannitol Salt Agar
NA Nutrient Agar
OD Optical density
CFU Colony Forming Unit
Hrs Hours
Fig. Figure
et.al., And others
Conc. Concentration
°C Degree Celsius
g Gram
g/l Gram per liter
ml Milliliter
spp. Species
μg Microgram
μl Microliter
% Percent
nm Nanometer
mm Millimeter
lb Pound
 7

LIST OF TABLES

Table No. Topic Page No.

3.1 Combination and concentration of plant materials used for the growth of E.
coli.

3.2 Combination of plant materials evaluated for the growth of E. coli, S. aureus
and Streptococcus spp. (environmental isolate).
4.1 Comparative O.D. values for E. coli in PBM1 of plant broth and nutrient
broth medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

4.2 Comparative O.D. values for E. coli in PBM2 of plant broth and nutrient
broth medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

4.3 Comparative O.D. values for E. coli in PBM3 of plant broth and nutrient
broth medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

4.4 Comparative O.D. values for E. coli in PBM4 of plant broth and nutrient
broth medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

4.5 Comparative O.D. values for E. coli in PBM5 of plant broth and nutrient
broth medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

4.6 Comparative O.D. values for E. coli in PBM6 of plant broth and nutrient
broth medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

4.7 Comparative O.D. values for E. coli in PBM7 of plant broth and nutrient
broth medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

4.8 Comparative O.D. values for S. aureus in PBM7a of plant broth and
nutrient broth medium after 24 hrs, 48 hrs and 72 hours of incubation at
600nm.

4.9 Comparative O.D. values for S. aureus in PBM7b of plant broth and
nutrient broth medium after 24 hrs, 48 hrs and 72 hours of incubation at
600nm.

4.10 Comparative O.D. values for Streptococcus spp. in PBM7a of plant broth
 10

CHAPTER-1
INTRODUCTION
 17

 Present a viscosity, osmotic pressure and pH that are all close to the ideal range.
There are many different types of culture media, and these media can be categorized into several
groups based on their chemical makeup, physical characteristics, intended application or
production process. The main types of media that are mostly used in the laboratories they are;
Basal media: The term "basal media" refers to media that can be utilized for the growth of
bacteria without the need for media enrichment. Examples include peptone water, nutrient broth
and nutrient agar. In this medium, Staphylococcus and Enterobacteriaceae flourish.
Enriched media: The media are enriched usually by adding blood, serum or egg. Blood agar
and Lowenstein-Jensen media are two examples of enriched media. Blood agar media support
Streptococci growth.
Selective media: This media favor the development of desired bacteria while limiting the growth
of undesirable bacteria, these media promote the growth of a particular bacterium. MacConkey
agar, Lowenstein-Jensen media and tellurite media are a few examples (tellurite inhibits the
growth of most of the throat organisms except Diphtheria bacilli). For inhibition, an antibiotic
may be added to a medium.
Differential or Indicator media: Two types of microorganisms that are growing in the same
medium can be distinguished using a so-called "differential" or "indicator" culture medium. In
this kind of medium, the biochemical traits of a microorganism growing in the presence of
particular nutrients or indicators (like neutral red or methylene blue) added to the medium are
used to visually indicate the traits of a microorganism. MacConkey agar (produced by the
fermentation of lactose), mannitol salt agar (by the fermentation of mannitol) are the examples.
Transport media: These media are employed when specimens cannot be cultivated right after
collection. The sample isn't allowed to dry out while being transported and harmful bacterial
growth is also slowed down. Example of transport media are Cary-Blair agar, Amies agar
transport swabs (Erkmen, 2021).
On the basis of consistency, media can be categorized into three types: Liquid, Solid and Semi-
solid media;.
Liquid media: Nutrient broths, i.e., liquid media containing nutrients, are the most often used
growth media for microorganisms. Microorganisms often expand exponentially in such broths
until their growth is inhibited by an excess of growth-inhibiting chemicals or a lack of available
nutrients.
Solid media: On the other hand, solid media enable the development and isolation of certain
microbial colonies on their surface. A single colony forming unit (CFU), which can be a single
bacterium, is the source of every colony. Therefore, it is presumed that every cell in a colony
belongs to the same strain and species. Agar, a mixture of agarose and agaropectin of algal origin
that very few bacteria can degrade, is added between 1 and 2% while solid culture media are
being made (liquefy). The media are autoclaved, cooled to a temperature of roughly 45 °C, and
then placed into petri dishes where they harden and cool. The bacteria that are injected onto the
surface of the agar are given water and nutrients by the gelatinous mass.
 18

Semi-solid: The preparation of semisolid medium uses lower agar concentrations of 0.2% to
0.5%. They are used to cultivate microaerophilic bacteria or analyse bacterial motility by
cultivation in stab tubes and have a soft, custard-like consistency. Typhoid and colon bacilli can
be distinguished using semisolid medium. Agar plates that are semi-solid must be handled
upright (Fermando, 2012).
On the basis of convenience, media is divided into two categories;
Ready- to- use: The practical choice for microbiology labs is ready-to-use media. The solid
media are easily poured into Petri plates, slant tubes or bigger bottles, whilst the liquid media are
easily sterilized and distributed in plastic bags, tubes and bottles of different sizes. Before
placing the agar plates into bottles of agar media, it is important to re-melt the agar and chill it to
44–47 °C.
Dehydrated culture media: On the other hand, commercially available dehydrated culture
media are long-term stable form (up to 5 years), either powders or granules and contain all the
components of the growth media, with the exception of water. These dehydrated medium must
be dissolved in water and autoclaved before use in the microbiological laboratory.
On the basis of composition, media are mainly of two types;
General- purpose media: Microbiology laboratories regularly employ general-purpose media,
commonly referred to as basic or complex media, to develop a wide range of bacteria. They
differ significantly in composition from batch to batch because they contain complex organic
materials like yeast, animal or plant material that are not chemically accurately determined.
These medium are used to cultivate non-picky microorganisms that don't require additional
nutrients for growth. In the microbiological laboratory, general-purpose media such as nutrition
agar and broth, peptone water and tryptic soy broth and agar are frequently used.
Synthetic based media: On the other hand, synthetic media are made entirely of known
chemical constituents. These media are specifically used to research the physiology, metabolism
and dietary needs of specific bacteria.
On the basis of oxygen content, media can be;
Anaerobic growth media: Anaerobic bacteria can tolerate little to no oxygen, hence it may be
necessary to physically or chemically limit their medium. To remove the dissolved oxygen from
these media, boil them and add reducing agents such sodium thioglycolate. Sealing the media
with sterile liquid paraffin is a common method of preventing oxygen from entering during
incubation. The anaerobic metabolism of facultative anaerobes or microbes that grow aerobically
in the presence of oxygen but switch to fermentation in the absence of oxygen can also be
studied using anaerobic culture medium (Mohane, 2020).
All of the listed media are prepared in synthetic form, are expensive, difficult to find and are not
ecofriendly. Additionally, some of these media are based on animal products or might be
considered to have been taken from animals. The goal of the current study is to develop this kind
of media for the culture of various microorganisms that are entirely based on plants and plant
products. A number of plants and plant parts used in the study are Oryza sativa (Rice) seeds,
 19

Ficus carica (Fig) leaves, Musa acuminata (Banana) leaves, Glycine max (Soyabean) seeds and
Pisum sativum (Pea) seeds were selected on the basis of their content of nutrients that are
essential for the growth of microorganisms. Rice grains are rich in carbohydrates, fig leaves
contain various types of vitamins, soyabean rich in protein source and dietary fibres and pea
seeds contain minerals and vitamins. These plant materials were used in different combinations
according to their nutrition content in order to formulate media for the growth of E. coli, S.
aureus and Streptococcus spp. one environmental isolate. Potential of these plant based media
were evaluated as selective and general purpose media for the growth of microorganisms.
2.1 Oryza sativa (Rice)
Asian rice, also known as Oryza sativa, is a member of the Poaceae family of plants. It is a
monocot that grows as both a perennial and an annual plant in tropical climates.
The most extensively cultivated tropical crop is rice and more than 400 million tonnes of milled r
ice are produced annually. Since ancient times, people have understood the value of rice (Alford,
2003). In India, it was originally referred to as "dhanya," which means "the sustainer of the
human race. Due to the labor-intensive nature of rice farming and the need for a lot of water, it
is best suited for places with cheap labor costs and high rainfall. The utilization of water-
controlling terrace systems, however, allows rice to be produced almost anywhere, including on
a steep hill or mountain environment. Despite being a native of Asia and a few regions of Africa,
its parent species have spread around the world due to millennia of commerce and exportation.
According to estimates, 4% of the world's greenhouse gas emissions in 2010 came from the
production and consumption of rice. The plants grow to a height of about a metre, however
sometimes 6-9 meters fibrous root system. The hollow culm or stem of rice is made up of
internodes and nodes. The rice plant has sessile leaves. It is a semi-aquatic plant that can
transport oxygen from aerial portions to roots through the presence of arenchymatic cells or leaf
culm and roots (Laha, 2017 and Ito, 2004).
2.1.1 Nutritional
Rice is a nutritional staple food which provides instant energy as its most important components
is carbohydrate (starch). Rice starch is used to thicken laundry and as a cosmetic dusting powder.
Rice flour is rich in starch, calcium, protein, magnesium, phosphorus, iron and vitamins. Rice is
utilized in a wide range of crafts and religious rituals in addition to being consumed in a variety
of ways. It is also a component of medicines and cosmetics.
2.2 Ficus carica (Fig)
The family Moraceae includes Ficus carica. It is frequently referred to as a "fig." It is grown or
farmed commercially in locations where it is used as a crop because it is eaten as a fruit. It is a
fruit tree that originated in the Mediterranean region and western Asia. It has been domesticated
since ancient times and is today a popular ornamental plant that is grown all over the world. The
type species of the genus Ficus, which has approximately 800 tropical and subtropical plant
species, is Ficus carica. The fig is a significant crop that is primarily available in the
summertime throughout the world. Figs are a distinctive fruit with a peel that can be eaten in
either purple or green. The fruit's flesh is pink and has a delicate flavor. Fig leaves are huge, flat
 10

CHAPTER-1
INTRODUCTION
 11

CHAPTER-1 INTRODUCTION

Media contains all the nutrients which are necessary for the growth of microorganisms including
nitrogen source, carbon source, energy, growth stimulants and few trace elements. A substance
that promotes the growth and survival of microorganisms is known as a microbiological culture
medium. Components of culture media include nutrients, growth-promoting substances, energy
sources, buffer salts, minerals, metals and gelling agents (for solid media). Culture media contain
all the elements that most bacteria need for growth and survival, so these media are used for the
general cultivation and maintenance of bacteria kept in laboratory culture collections.
Microorganisms can't be cultured with just one kind of culture medium because of their diverse
nature, traits, habitats, and dietary needs (Shareef, 2019). Growth medium used to grow bacteria
is called bacterial culture media. Bacterial culture media could either be general purpose media
or selective media. General purpose media is used to grow the majority of organisms because
they don't have any growth inhibitors. Examples of general-purpose media include blood agar
bases and standard agar. The majority of non-fastidious bacteria are supported on simple media
called basal media e.g., peptone water, nutrient agar, and nutrient broth. However, selective
media is used when only a particular type of bacteria needs to be cultivated. As a result, the
majority of selective culture medium used for bacterial growth contains complicated chemicals
that ultimately raise the product’s value. The practical testing of microbe is more challenging
when educational institutions lack sufficient resources for purchasing such type of medium. As a
result, an alternate medium that is straightforward in composition, inexpensive and widely
accessible must be developed (Bonnet et al., 2019).
Microbial culture media may be liquid, solid or semi-solid on the basis of their consistency.
(Srijoni et.al., 2015). Agar or gelatins are solidifying agents used in solid and semi-solid media.
Liquid media does not contain any solidifying agents. Examples of liquid media are broths e.g.
nutrient broth is most commonly used liquid media. Liquid media are available for use in test-
tubes, bottles or flask and bacteria growth in these media is observed as general turbidity.
Microorganisms in these broths proliferate exponentially until their ability to reproduce is
restricted by an inadequate supply of nutrients or a buildup of chemicals. Solid media are
prepared by adding 1-2% agar. It allows the growth and isolation of individual microbial
colonies on their surface. Every colony has a single colony-forming unit, which may be single
bacteria. As a result, it is presumed that every cell in a colony belongs to the same species and
strain. Examples of solid media are: nutrient agar, blood agar, MacConkey agar. Nutrient agar
media is frequently employed as a general-purpose medium because it contains many nutrients
needed for the growth of microorganisms. Nutrient agar is popular because it can grow a variety
of bacteria and fungi. This medium composed of beef extract, yeast extract, sodium chloride,
agar and peptic digest of animal tissue (Uthayasooriyan, 2016). However, semi- solid media are
prepared with lower agar concentration of 0.2- 0.5%. These media are fairly soft, useful in
demonstrating bacterial motility and could differentiate between motile and non- motile strains.
Typhoid and colon bacilli can be distinguished using semisolid medium. Examples of semi- solid
media are- Hugh and Leif son’s oxidation fermentation medium, Stuart’s and Amies media and
mannitol motility media etc. The microbiologist uses a variety of culture media for various
purposes e.g., isolating growth, maintaining pure cultures, identifying microorganisms based on
 12

their biochemical and physiological characteristics, testing for microbial contamination,

checking for antimicrobial agents, determining viable count and determining antibiotic
susceptibility (Ugah, 2018). In the pharmaceutical, food and beverage industries, culture media
continue to be the gold standard for counting and identifying microorganisms.
Currently there are thousands of distinct media types that can be broadly categorized into two
main groups: chemical media and organic media. Chemical defined medium is a medium in
which all the chemical composition used are known. Components of chemically defined media
include lipids, recombinant insulin or zinc, recombinant transferrin or iron, selenium and an
antioxidant thiol like 2-mercaptoethanol or 1-thioglycerol etc. There are number of bacteria
which require organic material for their growth and such types of microorganisms receives the
nutrients from organic medium (Jadhav, 2018). Chemically defined media used for bacterial
growth contain complex components or sometimes animal based products which ultimately raise
the product’s value. There are some media that contain materials derived from animals e.g.,
sheep or blood agar. Blood agar that contains 0.3% beef extract and Sheep blood agar contains
5% sheep blood, casein and yeast extract. These animal-based media are expensive and
occasionally hard to find. These media may result in allergies, immunosuppression and other
illnesses. Sometimes laboratory workers are adversely affected by these animal-based media
(Yao et al., 2017). There are some media which require addition of serum which further
facilitate high risk of mycoplasma, fungus and viral infections. Some of the growth-inhibiting
substances that serum may also contain prevent the growth and proliferation of cultured cells. On
the other hand, alternative organic media which contains only plant and plant products does not
adversely affect the laboratory workers. Currently scientists and researchers are also moving
towards organic media in laboratory personnals. Earlier various plant-based media have already
been reported for culturing of microorganisms. Examples of such plant-based media are
Lycopersicon esculentum (tomato) juice agar, Allium cepa (onion) agar media, Beta vulgaris
(sugar beet) agar media, Pisum sativm (pea) agar media, Pinus roxberghii (pine) agar media,
Lowsonia intermis (henna) agar media, Rhus cotinus (smoke tree) agar media, Brassica
campestris (mustard) agar media and tobacco agar for the growth of fungi (Aigbogun et al.,
2018, Bharti et.al., 2013, Pant et al., 2012 and Khan et.al., 2004). The present study has been
designed by keeping these points in mind. This research aims to formulate general purpose
media for the cultivation of wide varieties of microorganisms as well as selective media for the
isolation of E. coli. The goal of this in-vitro study is to replace the commercial ones with less
expensive, readily available and less time-consuming media. The available synthetic media are
less sensitive and are of high cost. The objective of this research is to formulate media for the
growth of bacteria found on our natural domain. Himachal Pradesh has a vast diversity of plant
life due to varied agro-climatic condition which ranges from subtropical to extremely cold.
Natural media made from organic materials can serve as an alternative to currently used
conventional media. Natural media are partly or fully composed of natural materials such as
plants or plant products. These media are inexpensive, easily accessible and easy to prepare. In
this present study, plant-based media will be prepared by using Oryza sativa (Rice) seeds, Ficus
carica (Fig) leaves, Musa acuminata (Banana) leaves, Glycine max (Soyabean) seeds and Pisum
sativum (Pea) seeds. These media could offer a novel alternative for currently available synthetic
media for the cultivation of variety of bacteria.
 18

Semi-solid: The preparation of semisolid medium uses lower agar concentrations of 0.2% to
0.5%. They are used to cultivate microaerophilic bacteria or analyse bacterial motility by
cultivation in stab tubes and have a soft, custard-like consistency. Typhoid and colon bacilli can
be distinguished using semisolid medium. Agar plates that are semi-solid must be handled
upright (Fermando, 2012).
On the basis of convenience, media is divided into two categories;
Ready- to- use: The practical choice for microbiology labs is ready-to-use media. The solid
media are easily poured into Petri plates, slant tubes or bigger bottles, whilst the liquid media are
easily sterilized and distributed in plastic bags, tubes and bottles of different sizes. Before
placing the agar plates into bottles of agar media, it is important to re-melt the agar and chill it to
44–47 °C.
Dehydrated culture media: On the other hand, commercially available dehydrated culture
media are long-term stable form (up to 5 years), either powders or granules and contain all the
components of the growth media, with the exception of water. These dehydrated medium must
be dissolved in water and autoclaved before use in the microbiological laboratory.
On the basis of composition, media are mainly of two types;
General- purpose media: Microbiology laboratories regularly employ general-purpose media,
commonly referred to as basic or complex media, to develop a wide range of bacteria. They
differ significantly in composition from batch to batch because they contain complex organic
materials like yeast, animal or plant material that are not chemically accurately determined.
These medium are used to cultivate non-picky microorganisms that don't require additional
nutrients for growth. In the microbiological laboratory, general-purpose media such as nutrition
agar and broth, peptone water and tryptic soy broth and agar are frequently used.
Synthetic based media: On the other hand, synthetic media are made entirely of known
chemical constituents. These media are specifically used to research the physiology, metabolism
and dietary needs of specific bacteria.
On the basis of oxygen content, media can be;
Anaerobic growth media: Anaerobic bacteria can tolerate little to no oxygen, hence it may be
necessary to physically or chemically limit their medium. To remove the dissolved oxygen from
these media, boil them and add reducing agents such sodium thioglycolate. Sealing the media
with sterile liquid paraffin is a common method of preventing oxygen from entering during
incubation. The anaerobic metabolism of facultative anaerobes or microbes that grow aerobically
in the presence of oxygen but switch to fermentation in the absence of oxygen can also be
studied using anaerobic culture medium (Mohane, 2020).
All of the listed media are prepared in synthetic form, are expensive, difficult to find and are not
ecofriendly. Additionally, some of these media are based on animal products or might be
considered to have been taken from animals. The goal of the current study is to develop this kind
of media for the culture of various microorganisms that are entirely based on plants and plant
products. A number of plants and plant parts used in the study are Oryza sativa (Rice) seeds,
 20

and broad, measuring 12–25 cm in length and 10–18 cm in breadth on average. The leaf's 3.5
lobes have deep veins and noticeable steam and it is a bright, vibrant green colour. Fig leaves
smell quite good (Veberic et al., 2016).
Figs and their leaves are nutrient-rich and provide a range of health advantages. They may aid in
maintaining healthy digestion, lowering the risk of heart disease and controlling blood sugar
levels (Crisosto et.al., 2011).
Fresh or dried, figs play a significant role in the Mediterranean diet. They are particularly high in
fibre, trace minerals, polyphenols, proteins and carbohydrates. Figs can be consumed fresh,
dried, or processed into delicacies like jam, rolls and biscuits. The majority of commercial
production is in dried and processed forms since ripe fruit is difficult to transport and keep fresh.
About 80% of a raw fig's weight is made up of water, 20% of it of carbohydrates, and very little
protein, fat or micronutrients. They provide a fair amount of dietary fiber. In 2018, there were
1.14 million tones of raw figs produced worldwide, with 64% of the total coming from Turkey
and the North African nations of Egypt, Morocco and Algeria.
2.2.1 Nutrition
Figs and their leaves are rich in nutrients and may provide a number of health advantages. They
could help manage blood sugar levels, encourage good digestion and lower the risk of heart
disease. Additionally, figs have trace amounts of numerous minerals, but they are especially high
in copper and vitamin B6. The creation of blood cells, connective tissues and neurotransmitters,
as well as metabolism and energy production, are all supported by the essential mineral copper.
2.3 Glycine max (Soybean)
Glycine max, sometimes called soya bean. It is an annual legume from the Fabaceae family of
peas. The cultivated soybean is the species that is most popular Glycine max. The soybean's
natural area is in East Asia, however the majority of the species are restricted to Australia.
Several species range from East Asia to Australia (e.g., G. tomentella and G. tabacina). The seed
can be eaten. It is an upright branching plant with a height of more than two meters. Yellow,
green, brown, black or bicolor seeds are all possible (Badole et.al., 2012).
2.3.1 Nutrition
It is among the most abundant and affordable sources of protein. 50 percent of the seed's meal,
which is made up of 63 percent meal and 17 percent oil, is protein (Carlos et al., 2020). The
soybean is the most significant bean commercially since it provides ingredients for hundreds of
chemical goods and millions of people with vegetable protein. In many regions of the world,
people and animals eat a lot of soybeans since they are one of the richest and least expensive
sources of protein (Modgil et.al., 2021). Phytic acid, dietary minerals and B vitamins are all
present in substantial quantities in soybeans. Another byproduct of processing the soybean crop
is soy vegetable oil, which is used in both food and industrial uses. The most crucial source of
protein for feed for agricultural animals is soy (that in turn yields animal protein for human
consumption).
2.4 Musa acuminata (Banana)
 21

Commonly referred to as "Apple of Paradise" and generally called "banana." It is a member of

the family Musaceae (Morton, 1987). The largest herbaceous blooming plant is the banana plant.
In tropical areas, there are numerous types of banana plants that can reach heights of up to 7-8
meters. Its leaves are now used as a common cooking ingredient. A stalk (petiole) and a blade
make up the leaves of banana plants (lamina). They have 8–12 leaves per tree and are broad,
elongated and massive, measuring an average of 2 meters in length and half a meter in width.
The leaves surfaces are pliable and waxy and they range in color from light to dark green.
Bananas used in cooking may be referred to as "plantains" in some nations to distinguish them
from dessert bananas. The fruit's shape, colour and firmness vary, but it is often elongated and
curved. It has soft flesh that is high in starch and is coated in a rind that, when mature, can be
green, yellow, red, purple or brown. Near the top of the plant, clusters of fruits grow upward.
Musa acuminata and Musa balbisiana are two wild species from which almost all edible
seedless (parthenocarp) bananas today are derived. Depending on their chromosomal makeup,
the majority of cultivated bananas go by the scientific names Musa acuminata, Musa balbisiana
and Musa paradisiaca for the hybrid Musa acuminata M. balbisiana. Musa sapientum, the
previous scientific name for this hybrid, is no longer used (Ploetz, 2015).
Native to Australia and tropical Indomalaya, Musa species are thought to have initially been
domesticated in Papua New Guinea. They are cultivated in 135 nations mostly for its fruit
however, they are also produced for their fiber, wine, beer and aesthetic purposes. In 2017, India
and China produced the most bananas in the world, jointly making up about 38% of the overall
production.
2.4.1 Nutrition
Bananas are a good source of potassium, fibre, vitamin B6, vitamin C and a number of
phytonutrients and antioxidants. The majority of the carbohydrates in bananas are found as
sugars in ripe bananas and as starch in unripe bananas. In addition to being a good source of
minerals like sodium and potassium, banana leaves also contain polyphenols, which are natural
antioxidants (Mariya et al., 2020).
2.5 Pisum sativum (Pea)
The little spherical seed or seed-pod of the pod fruit Pisum sativum is most frequently referred to
as the pea. There are numerous green or yellow peas in each pod. Pea pods are considered to be
fruit by botanists since they contain seeds and grow from the ovaries of a (pea) flower. Other
edible seeds from the Fabaceae family, including the pigeon pea (Cajanus cajan), the cowpea
(Vigna unguiculata) and the seeds from other species of Lathyrus, are also referred to by this
name. The pea or Pisum sativum, often known as garden pea, is a herbaceous annual plant in the
Fabaceae family that is practically grown everywhere for its tasty seeds. Fresh, canned or frozen
peas are all readily available, and dried peas are frequently used in soups. The edible pods of
some kinds, such as sugar peas and snow peas, are consumed raw or cooked like green beans and
are commonly used in East Asian cuisines. The seeds are a good source of protein and dietary
fiber and the plants are not too difficult to grow.
 22

The pea plant is a tough leafy perennial that may grow up to 1.8 meters (6 feet) in length and has
hollow stems that trail or climb. The stems contain compound leaves with three pairs of leaflets
and have terminal tendrils to aid in climbing. Two to three reddish purple, pink or white
butterflies-shaped flowers are produced on each stem. The fruit is a pod that matures to a length
of 10 cm (4 inches) before breaking in half. Five to ten seeds are linked by thin stalks inside the
capsule. Green, yellow, white or variegated seeds are available.
2.5.1 Nutrition
Peas are rich in lutein, dietary fibre, protein, vitamins and minerals. About one-fourth of dry
weight is made up of sugar and one-half of it is protein (Jegtvig et al., 2007). Compared to
glutathione, pea seed peptides are less effective in neutralising free radicals, but they are more
effective at chelating metals and preventing the oxidation of linoleic acid (Pownall et al., 2010).
Pea and lentil allergies exist in certain people (Monge et al., 2004).
Synthetic media used for the growth of E. coli, S. aureus and Streptococcus spp. are nutrient
agar. EMB (Eosin Methylene Blue) selective media used for the growth of E. coli and this
organism grow in this media formed metallic green sheen. Selective media used for the growth
of S. aureus is MSA (Mannitol Salt Agar). A number of artificial media are used for growing
different types of bacteria. But these media are costly and require intensive labour to produce
them. Their are studies which reports that these plant based media are better option to replace the
synthetic media. Plant based culture media i.e. Tomato juice agar was used to observe ascosporic
formation, was combined with niger seed agar, casein agar and sunflower seed agar to
differentiate C. dubliniesnsis and C. albicans (Alves, 2006). Pant et.al., 2012 reported that plant
based culture media prepared from henna leaf, mustard seed and smoke tree leaf based media
supported the best growth of Cryptococcus neoformans. Another study on evaluation of plant
based liquid and solid media reveals that the Allium cepa (onion), Lycopersicon esculentum
(tomato), Beta vulgaris (sugar beet) and Pisum sativum (pea) based culture media were best
suited for the recovery of Candida albicans during 72hrs of incubation, but they observed best
recovery in tomato agar, sugar beet agar and onion agar at different concentrations (Bharti et.al.,
2013). Youssef et.al., 2015 reported that plant based culture media prepared from juices of
Opuntia ficus- indica and Aloe vera and sap of Aloe arborescens were rich enough to support the
growth of rhizobacteria. Elhussein et.al., 2018 also reported that K. oxytoca exhibited very good
growth in liquid culture media prepared from slurry homogenates and the powders of clover and
cactus.
Future outcome
The present study has been designed to formulate plant based selective and general purpose
media by using plants and plant parts i.e. Oryza sativa (Rice) seeds, Ficus carica (Fig) leaves,
Musa acuminata (Banana) leaves, Glycine max (Soyabean) seeds and Pisum sativum (Pea) seeds.
These plant based media is simple, natural, easy available, cost- effective and easy to use as
compared to synthetic media, which are more complex and expensive. These media are more
ecofriendly, less harmful to users as there are very less chance of occurrence of any allergen in
these media as they are prepared from natural products. Further, disposal of media prepared from
plant and plant products are easy as compared to synthetic media. Therefore the present study
 23

has been designed by keeping these points in mind. Although there are very less studies on
formulation of plant based media and further studiesare required in order to produce such media
on large scale.
 30

The ability of an organism to move by itself is called motility. This test is done to determine if an
organism is motile or non-motile. Semi solid medium was inoculated with a straight wire about
8-10mm deep into the medium only once and incubated at 37°C for 24 hrs. Diffused growth
from the stab line showed positive motility test whereas growth restricted to stab line reflects the
bacteria species is non motile.
Carbohydrate fermentation test
This test determines the ability of an organism to degrade and ferment carbohydrates (glucose,
lactose, mannitol etc.) with production of an acid or acid and gas. Four tubes containing four
different kinds of broth namely glucose, lactose, and mannitol which consist of 1% sugar, 1%
peptone water and 1% Andrade’s reagent were taken. Inverted Durham tube placed in each tube
was in inverted position. Autoclaving was done at 10 lb. pressure. Each tube was inoculated with
bacterial culture and incubated at 37°C for 24 h. Pink color formation indicated acid production
while cream or yellow color with slight pink tinge showed no fermentation of carbohydrates.
Production of pink color and accumulation of gas in Durham tube indicated generation of both
acid and gas. Paragenesis a non-fermenter of glucose while certain other bacteria Escherichia
coli capable of fermenting glucose.
Indole production test
This test determines the ability of an organism to split indole from amino acid tryptophan and is
generally performed to differentiate species of the family Enterobacteriaceae. In this test, 24h
cultures in peptone water were incubated at 37°C. Kovac’s reagent (4-5 drops) was added to it.
The production of red color indicates the production of indole from tryptophan in a positive case.
Methyl red and Voges-Proskauer test
Methyl red (MR) and Voges-Proskauer (VP) tests are generally used in the biochemical
identification of bacterial species. These tests are based on the detection of specific breakdown
products of carbohydrate metabolism. MR/VP broth in a volume of 5 ml taken in test tubes and
sterilized by autoclaving at 15 lb pressure (121°C) for 15 min. Test isolates were inoculated in
test tubes containing MR/VP broth and incubated 37°C for 24 hrs. Four to five drops of MR/VP
reagents were added to 24hrs old MR/VP culture. The appearance of red or pink color indicated
a positive reaction.
Citrate utilization test
Citrate utilization test determines capability of an organism to utilize citrate as the sole source of
carbon for their metabolism. Simmons’s citrate medium was prepared by dissolving 2.42 grams
of powder in 100ml of distilled water and autoclaving was done at 121°C for 15 minutes. Slants
were prepared and test bacterial colonies were streaked over the surface of the slants. Incubation
was done at 37°C for 24 hrs. The production of blue color indicated positive test.
Urease test
Urease test was done to determine the ability of an organism to hydrolyze urea by the action of
urease enzyme. Urea agar base (2.40 grams) was added in 95 ml of distilled water. The medium
 31

was heated to dissolve the medium completely and sterilized by autoclaving at 10 lb pressure for
20 minutes followed by cooling to 50°C. Sterile 40% urea (5 ml) solution was added and mixed
well. Sterile tubes were dispensed with medium and allowed to set in the slanting position.
Overheating or reheating of the medium was avoided as urea decomposed very easily (Zhou X
et.al., 2015)
3.4 Preservation of the strains
The strains of E. coli, S. aureus and isolate of Streptococcus spp. were maintained on slants of
EMB agar media, MSA agar media and nutrient agar media (Hi-media, Mumbai). Slants were
preserved in 10% glycerol at -20°C then subculturing was done on regular basis in order to
maintain the fresh cultures for the experiment and their composition are given in Annexure- 2
(Abdullahi, 2020).
3.5 Preparation of plant-based media
All combinations of plant-based media (Table 3.1) were evaluated for the growth of E. coli
MTCC 1687 by following the method of Elsawey et.al., 2020. Dipotassium hydrogen phosphate
was used as selective agent for the growth of E. coli in PBM1 (Plant Based Media 1) and PBM2
of plant-based media.
3.5.1 Preparation of plant-based liquid media
All plant based liquid media were prepared from selected combinations of plant materials (Table
3.1). The weighed amount of fine powder of respective plant materials with different
concentrations were added to 100 ml of distilled water and boiled for 10 minutes with
occasionally shaking. After that, media were filtered through Whatman No. 1 filter paper and the
filtrate and were autoclaved at 121°C under 15 lb pressure for 15 minutes. In sterile test tubes,
15-10 ml of filtrate of each combination was added separately.
3.5.1.1 Preparation of cell inoculum for plant-based liquid media
An inoculum was prepared by adjusting the turbidity to McFarland standard. 100µl inoculum
containing 104 cfu/ml of E. coli was added in plant broths prepared by using PBM1, PBM2 and
PBM3 of plant material. Similarly, 100µl inoculum containing 102 cfu/ml of E. coli was added in
PBM4, PBM5, PBM6 and PBM7 prepared from plant broths (Table 3.1). All culture media were
incubated at 37°C for 24hrs, 48hrs and 72 hours. At 600 nm, the optical densities were measured.
3.5.2 Preparation of plant based solid media
All selected combinations of plant based solid media for the growth of E. coli (Table 3.1) were
prepared by following the method of Hansen et.al., 2016. The measured amounts of fine
dehydrated powder of respective plant materials were added to 100 ml distilled water and the
mixture were then cooked for 10 minutes while being regularly stirred. The media were then
filtered through a muslin cloth to remove any large debris followed by filtration through
Whatman No. 1 filter paper. 1.3% agar were added to media and sterilized in an autoclave at
121°C for 15 minutes under 15 lb pressure after being heated to dissolve them. Approximately
 32

15-20 ml of media was added to sterile petri dishes separately, resulting in a 4 mm thickness in
the plates.
3.5.2.1 Preparation of cell inoculum for solid media
0.5mL of the cell suspension was taken in an eppendorf tube. 100µL of cells and 400µL of 0.4%
Trypan Blue (final concentration: 0.32%) were mixed in a fresh eppendorf tube and mixed
gradually. 100 µL of the Trypan Blue-treated cell suspension were applied to the hemocytometer
chamber. Cell suspension distributed evenly to the hemocytometer chamber by allowing
capillary action. Hemocytometer's grid lines were concentrated with the help of a microscope.
The number of active cells was counted in each of the 16 squares. Only cells that were inside the
square and those that were on the bottom or right boundary line were counted. Once all 4 sets of
16 corners have been counted, move the hemocytometer to the following square with 16 corners
and continue counting. 91 cells of E. coli for PBM1 and PBM2, 88 cells for PBM3, PBM4 and
PBM5 and 97 cells for PBM6 and PBM7 were inoculated on the plant-based agar medium in
order to determine the actual cfu (colony forming unit) recovered after 24hrs, 48hrs and 72 hours
of incubation.
3.5.3 Spread plate method
Using a sterile L-shaped spreader, 100µl of inoculum containing 91 cells i.e., 9.1× 104 cfu/ml in
PBM1 and PBM2, 88 cells 8.8×102 cfu/ml in PBM3 and PBM4 and 97 cells i.e. 9.7×104 cfu/ml
in PBM5, PBM6 and PBM7 was spreaded over the surface of all plant based solid media. The
plates were incubated for 24h, 48h and 72 hours at 37°C. Numbers of colonies were counted
using a colony counter after being incubated.
Combination showing best growth was further evaluated for their potential as plant based general
purpose media (Table 3.2). The best combination supporting the growth of E. coli was further
evaluated for the growth of other microbes e.g., S. aureus and Streptococcus spp. (Table 3.2)
which was an environmental isolate by following the same method as described above under
section 3.5.
3.6 Phytochemical analysis of plant materials
The plant materials were analyzed to check the nature of chemical constituents such as alkaloids,
saponins, flavonoids, reducing sugar, tannins, glycosides, terpenoids, coumarins and protein.
Test for alkaloids
A few drops of Meyer’s reagent were added to 1ml of each plant material. Development of
yellow color indicated the presence of alkaloids (Usman, 2009).
Test for saponins
1ml of each plant material was added in 1ml of distilled water and shake it vigorously then leave
the test tubes for 5 minutes. Foam formation indicated the presence of saponins.
Test for flavonoids
 33

2ml of each plant material was added in test tubes, 2% NaOH (sodium hydroxide) was added
drop wise drop and then intense yellow color appeared. Finally, with the addition of dil. HCl and
then color disappear. It indicated the presence of flavonoids (Mathew et al., 1989).
Test for reducing sugar
1ml of each plant material was added in 1ml of distilled water five to eight drops of freshly
prepared Fehling A and Fehling B solutions were added. Appearance of brick red precipitate
indicated presence of reducing sugar.
Test for tannins
Few drops of 10% lead acetate were added in 1ml of each plant material. Presence of tannins was
observed by occurrence of black precipitate.
Test for glycosides
1ml of each plant material was dissolved in 1ml of glacial acetic acid containing one drop of
ferric chloride FeCl3 solution. The mixture was then poured into a test tube containing 1ml of
conc. H2SO4. A brown ring at the interphase indicated the presence of glycoside in the selected
plant material (Usman et al., 2009).
Test for terpenoids
1ml of each plant material was dissolved in 2ml of chloroform and evaporated to dryness. 2ml of
conc. H2SO4 was then further added to it and heated for about 2 min. Development of greyish
color indicated the presence of terpenoids.
Test for coumarins
1ml of ethanol- KOH solution was added in 1ml of each plant material and appearance of yellow
precipitates indicated positive test for coumarins.
Test for protein test
1ml of each plant extract was added to 4% sodium hydroxide and few drops of 1% CuSO 4
solution were added. Appearance of violet or pink color indicated the presence of proteins in
selected plant materials (Bruck et al., 2020).
Table 3.1 Concentration of plant materials used for the growth of E. coli

Plant Combination used Concentration used

Based
Media

PMB1* Rice+ Fig+ Soyabean+ 0.25g+0.25g+0.5g+0.2g+0.5g +

Dipotassium hydrogen 100ml
 34

phosphate+ NaCl +
Distilled Water

PBM2* Rice+ Fig+ Soyabean+ 0.5g+0.25g+0.5g+0.2g+0.5g+100ml

Dipotassium hydrogen
phosphate+ NaCl+
Distilled Water
PBM3* Rice+ Fig+ 0.25g+0.25g+0.5g+0.5g+100ml
Soyabean+NaCl +
Distilled Water
PBM4* Rice+ Fig+ 0.5g+0.25g+0.25g+0.5g+100ml
Soyabean+NaCl+
Distilled Water
PBM5* Rice+ Fig+ Banana+ 0.5g+0.25g+0.25g+0.5g+0.5g+100ml
Soyabean+NaCl+
Distilled Water
PBM6* Rice+ Fig+ Pea 0.25g+0.25g+0.25g+0.5g+0.5g+
+ Soyabean+NaCl+ 100ml
Distilled Water
PBM7* Rice+ Fig+ Pea 0.5g+0.25g+0.25g+0.5g+0.5g+100ml
+ Soyabean+NaCl+
Distilled Water

* 1.3gm agar used for the preparation of plant based solid media.
Table 3.2 Combination of plant materials evaluated for the growth of E. coli, S. aureus and
Streptococcus spp. (environmental isolate).
Combination Plant Based Media Concentration
Rice+ Fig+ Pea PBM7a 0.25g+0.25g+0.25g+0.5g+0.5g+
100ml
+Soyabean+NaCl+
Distilled Water* PBM7b 0.5g+0.25g+0.25g+0.5g+0.5g+
100ml

* 1.3 gm agar powder added for the preparation of plant based solid media.
 35

CHAPTER-4
RESULTS AND DISCUSSION
 36

Chapter-4 Results and Discussion

E. coli, S. aureus and Streptococcus spp. (environmental isolate) were reconfirmed on the basis
of Gram’s staining and biochemical characterization.

4.1 Measurement of optical densities of plant based liquid media

Optical density (O.D) values of plant broths prepared from all combinations presented in table
4.1 (Fig. 4.1). Growth in terms of O.D value was found to be high in plant broths prepared from
PBM1 and PBM7 as compared to standard media used whereas growth in plant broths prepared
from PBM2, PBM3, PBM4 and PBM6 showed comparatively less growth when compared to
standard media. However, plant broths prepared from PBM5 showed comparable growth to
standard media. First two combinations (PBM1 and PBM2) mentioned in the table 3.1 were
evaluated for selective growth of E. coli as this media contain dipotassium hydrogen phosphate
as selective agent. A constant increase was found in O.D values of plant broths. It is evident
from the data that all the plant broths supported the growth of E. coli during 24hrs, 48hrs and 72
hrs of incubation. Maximum increase in O.D. values occurred after 48hrs and 72hrs of growth.

4.2 Recovery of E. coli on plant based solid media

Recovery of E. coli in colony forming unit (cfu) after 24hrs, 48hrs and 72 hrs of incubation are
presented in table 4.12. The recovery of the organism was seen with all the measured amounts
of the plant material and the recovery was comparable with standard media used (NA, EMB)
but the best recovery was observed in PBM7 (rice, fig, pea and soyabean agar media at 0.5g,
0.25g, 0.25g and 0.5g concentration) (Fig. 4.7). First two combinations (PBM1 and PBM2)
were represented in table 3.1 used as selective media does not support the growth of E. coli after
24hrs, 48hrs and 72 hrs of incubation suggesting that any component of media may act as
inhibitory agent for the growth of E. coli.

4.3 Recovery of other microorganisms on plant based media

The plant based solid media (rice, fig, pea and soyabean agar media) having best recovery rate
for E. coli was further evaluated for the growth of other microorganisms such as S. aureus and
Streptococcus spp. The selected plant based medium has shown higher growth in terms of O.D.
value and cfu/ml. Both isolates were best recovered from plant-based media prepared from rice,
fig, pea and soyabean at 0.5g, 0.25g, 0.25g and 0.5g concentration. Growth of these
 37

microorganisms on plant based media showed that these media could be used as general
purpose media for the growth of microorganism.

4.4 Phytochemical constituents

Phytochemical analysis was done for the selected plant material to check the nature of chemical
constituents such as alkaloids, saponins, flavonoids, reducing sugar, tannins, glycosides,
terpenoids, coumarins and protein. The presence or absence of constituents is represented in the
table 4.15. Rice has high content of saponins, terpenoids and protein test. Fig leaves are rich in
alkaloids, saponins, flavonoids, terpenoids and coumarins. However, banana leaves has high
content of alkaloids, saponins, flavonoids, reducing sugar, tannins, terpenoids, coumarins and
pea (seeds) are rich in flavonoids, glycosides, terpenoids and proteins. Soyabean seeds are rich
in flavonoids, tannins, glycosides and protein.

Table 4.1: Comparative O.D. values for E. coli in PBM1 of plant broth and nutrient broth
medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

Time period Plant broth Nutrient broth

(O.D) (O.D) (Standard
media)
PMB1 24 hours 1.516 1.484

48 hours 1.649 1.829

72 hours 2.333 1.885

 38

PBM1
2.5 2.333

Optical Density(O.D) 2 1.829 1.885

1.649
1.5161.484
1.5

Plant broth
1
Nutrient broth
0.5

0
24 hours 48 hours 72 hours
Time Period

Figure 4.1: Comparative O.D. values for E. coli in PBM1 of plant broth and nutrient broth
medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

Table 4.2: Comparative O.D. values for E. coli in PBM2 of plant broth and nutrient broth
medium after 24 hrs, 48 hrs and 72 hours of incubation at 600nm.

Time period Plant broth Nutrient broth

(O.D) (O.D) (Standard
media)
PMB2 24 hours 0.925 1.484

48 hours 1.119 1.829

72 hours 1.511 1.885

 45

PBM7a
1.8 1.636
1.6 1.512
1.4 1.273

Optical Density(O.D)
1.2 1.162
1.06
0.973
1
0.8 Plant broth
0.6 Nutrient broth
0.4
0.2
0
24 hours 48 hours 72 hours
Time Period

Figure 4.8: Comparative O.D. values obtained for S. aureus in PBM7a of concentration of
plant broth and nutrient broth medium after 24 hrs, 48 hrs and 72 hours of incubation at
600nm.

Table 4.9: Comparative O.D. values obtained for S. aureus in PBM7b of concentration of
plant broth and nutrient broth medium after 24 hrs, 48 hrs and 72 hours of incubation at
600nm.

Time period Plant broth Nutrient broth

(O.D) (O.D) (Standard
media)
PBM7b 24 hours 1.263 0.973
48 hours 1.718 1.060
72 hours 1.854 1.162
 46

PBM7b
2 1.854
1.8 1.718
1.6

Optical Density(O.D)
1.4 1.263
1.162
1.2 1.06
0.973
1
0.8 Plant broth
0.6 Nutrient broth
0.4
0.2
0
24 hours 48 hours 72 hours
Time Period

Figure 4.9: Comparative O.D. values obtained for S. aureus in PBM7b of concentration of
plant broth and nutrient broth medium after 24 hrs, 48 hrs and 72 hours of incubation at
600nm.

Table 4.10: Comparative O.D. values obtained for Streptococcus spp. in PBM7a of
concentration of plant broth and nutrient broth medium after 24 hrs, 48 hrs and 72 hours
of incubation at 600nm.

Time period Plant broth Nutrient broth

(O.D) (O.D) (Standard
media)
PBM7a 24 hours 1.728 1.399

48 hours 2.205 1.648

72 hours 2.396 1.809

 47

PBM7a
3

2.5 2.396
2.205

Optical Density(O.D)
2 1.728 1.809
1.648
1.399
1.5
Plant broth
1 Nutrient broth

0.5

0
24 hours 48 hours 72 hours
Time Period

Figure 4.10: Comparative O.D. values obtained for Streptococcus spp. in PBM7a of
concentration of plant broth and nutrient broth medium after 24 hrs, 48 hrs and 72 hours
of incubation at 600nm.

Table 4.11: Comparative O.D. values obtained for Streptococcus spp. in PBM7b of
concentration of plant broth and nutrient broth medium after 24 hrs, 48 hrs and 72 hours
of incubation at 600nm.

Time period Plant broth Nutrient broth

(O.D) (O.D) (Standard
media)
PBM7b 24 hours 2.116 1.399
48 hours 2.477 1.648
72 hours 2.535 1.809
 48

PBM7b
3
2.477 2.535
2.5
2.116

Optical Density(O.D)
2 1.809
1.648
1.399
1.5
Plant broth
1 Nutrient broth
0.5

0
24 hours 48 hours 72 hours
Time Period

Figure 4.11: Comparative O.D. values obtained for Streptococcus spp. in PBM7b of
concentration of plant broth and nutrient broth medium after 24 hrs, 48 hrs and 72 hours
of incubation at 600nm.

Table 4.12: Comparative colony forming units (cfu) recovered for E. coli on different
combinations of plant-based agar medium and standard media after 24 hrs, 48 hrs and 72
hours of incubation.
Plant Time Control Cfu recovered Standard Media
period on plant-based
based
media
media
used

Nutrient agar EMB

(Standard
media)
PBM1 24 Hrs No 224 >300 >300
growth
48 Hrs No 250 >300 >300
growth
72 Hrs No >300 >300 >300
growth
PBM2 24 Hrs No 256 >300 >300
 49

growth
48 Hrs No >300 >300 >300
growth
72 Hrs No >300 >300 >300
growth
PBM3 24 Hrs No No growth >300 >300
growth
48 Hrs No No growth >300 >300
growth
72 Hrs No 134 >300 >300
growth
PBM4 24 Hrs No 279 >300 >300
growth
48 Hrs No >300 >300 >300
growth
72 Hrs No >300 >300 >300
growth
PBM5 24 Hrs No 53 >300 >300
growth
48 Hrs No 65 >300 >300
growth
72 Hrs No 98 >300 >300
growth
PBM6 24 Hrs No >300 >300 >300
growth
48 Hrs No >300 >300 >300
growth
72 Hrs No >300 >300 >300
growth
PBM7 24 Hrs No 220 >300 >300
growth
48 Hrs No 289 >300 >300
growth
72 Hrs No >300 >300 >300
growth
 52

(c) Growth of S.aureus on nutrient agar media (d) Growth of S. aureus on MSA
(Mannitol Salt Agar) media
Figure 4.13 Colony forming unit (cfu) recovered for S. aureus on (a) Plant based media (b)
uninoculated control (c) Nutrient agar medium (d) MSA agar medium
Table 4.14: Comparative colony forming units (Cfu) recovered for Streptococcus spp. on
different combinations of plant-based agar medium and standard media after 24 hrs, 48
hrs and 72 hours of incubation.

Plant based Time period Control Cfu Nutrient agar

media used recovered on (Standard
plant based medium)
media
PBM7a 24 Hrs No growth >300 >300
48 Hrs No growth >300 >300
72 Hrs No growth >300 >300
PBM7b 24 Hrs No growth 115 >300
48 Hrs No growth 167 >300
72 Hrs No growth 238 >300
 53

(a) Colony forming unit (cfu) recovered for (b) Uninoculated control
Streptococcus spp. on plant based solid media

Figure 4.14 Colony forming unit (cfu) recovered for Streptococcus spp. on (a) Plant based
media (b) uninoculated control
Table 4.15: Phytochemical analysis of plant materials used

Rice Fig Banana Pea Soyabean

Alkaloids - + + - -
Saponins + + + - -
Flavonoids - + + + +
Reducing - - + - -
sugar
Tannins - - + - +
Glycosides - - - + +
Terpenoids + + + + -
Coumarins - + + - -
Proteins + - - + +
+ indicates presence

- indicates absence
 54

(a) (b) (c) (d) (e) (f) (g) (h) (i)

Figure 4.15 Phytochemical analysis of plant and plant products (a) Alkaloids (b) Saponins (c)
Flavonoids (d) Reducing sugar (e) Tannins (f) Glycosides (g) Terpenoids (h) Coumarins and (i)
Proteins
 55

Discussion

The present study on the evaluation of plant based broths of Oryza sativa (Rice) seeds, Ficus
carica (Fig) leaves, Musa acuminata (Banana) leaves, Glycine max (Soyabean) seeds and Pisum
sativum (Pea) seeds for supporting the growth of different types of bacteria with the various
concentration used that support the growth of E. coli, S. aureus and Streptococcus spp.
(environmental isolate) during 72 hrs of incubation.

This is an important observation in the direction of using such media for growing different types
of bacteria as a replacement for synthetic media such as Eosin Methylene Blue (EMB), Nutrient
agar (NA) and Mannitol Salt Agar (MSA) which are complex, not easily available and
expensive. Further studies are required to be done in this regard before arriving at definite
conclusion. These plant-based media can be utilized to support a variety of microbial growth
and their potential for supporting the development of commercially significant bacteria can also
be evaluated. Selected combination of plant-based media can be tested for the development of
other microorganisms as an alternative to the possibilities mentioned above.

Various researchers used the various plants in their study as a medium (Khan et.al., 2004;
Nandhakumar et.al., 2006). The confluent growth of Cryptococcus neoformans has been
discovered to be supported by plant-based media such as henna leaf-based media, mustard seed-
based media, and smoke tree leaf-based media, which makes them an excellent choice as
selective media for Cryptococcus neoformans (Pant et al., 2012). The distinction between C.
dubliniensis and C. albicans was made using tomato juice agar, a well-known medium used to
study ascosporic development, along with niger seed agar, casein agar and sunflower seed agar
(Alves et.al., 2016).

But the research on plant-based broth media that promotes the development of E. coli, S. aureus
and Streptococcus spp. has not been published. This is perhaps the first study which reports the
use of plant based broth and solid media supporting confluent growth of E. coli, S. aureus and
Streptococcus individually without any supplementation. Media including both synthetic and
natural are tested as general and selective media for the presumptive identification of E. coli, S.
aureus and Streptococcus spp. (environmental isolate). EMB (Eosin Methylene Blue) MSA
(Mannitol Salt Agar) and NA (Nutrient agar) are the three types of standard bacterial
 56

maintenance media. A common medium for the identification and cultivation of E. coli species is
EMB agar, which produces blue-black colonies with a green metallic sheen (Bharti et.al; 2013).
Whereas on MSA or NA, the colonies appear yellow or creamish white. Colonies appeared on
plant-based media are also creamish white in color.

In this study, five plant based media were tested for E. coli, S. aureus and Streptococcus spp.
(environmental isolate). The powdered form of all selected plants at their different
concentrations i.e., 0.25g and 0.5g in distilled water were used to prepare agar plates. Each plant-
based media was inoculated with E. coli, S. aureus and Streptococcus spp. observed after 72 hrs
for the growth and colony characteristics.

Oryza sativa (rice), Ficus carica (fig) and Glycine max (soyabean) agar selective based medium
(first andPBM2) for the cultivation of E. coli was light creamish in color and showed less
growth of E. coli as compared to standard medium (EMB) after 72hrs. Maximum increase in
O.D. value occurred after 48hrs or 72hrs of incubation.

Oryza sativa (rice), Ficus carica (fig) and Glycine max (soyabean) agar-based medium (third and
PMB4) was translucent, light white in color and showed good and countable growth after 72hrs
at all the concentrations. Maximum increase in O.D. value occurred after 72hrs of incubation.

Oryza sativa (rice), Ficus carica (fig), Musa acuminate (banana) and Glycine max (soyabean)
agar-based medium (PBM5) was white in color and showed growth of E. coli after 72 hrs at all
the concentrations. The colonies were creamish white in color and small in size as compared to
colonies that appeared on EMB and NA.

Oryza sativa (rice), Ficus carica (fig), Pisum sativum (pea) and Glycine max (soyabean) agar-
based medium (sixth and PBM7) was translucent, creamish white in color and showed good,
countable growth and heavy growth of E. coli after 72 hrs of incubation at all concentrations. So,
this media also seems to be better alternative for E. coli as compared to other standard media
(Khan et.al., 2004; Nandhakumar et.al., 2006 and Bharti et.al., 2013).

In PBM7 of plant based media was light white in color and showed good, countable growth of
other microorganisms i.e. S. aureus and Streptococcus spp. after 48hrs and 72hrs of incubation at
all concentrations. Maximum increase in O.D. value occurred after 48hrs or 72hrs of incubation.
. All plant-based media were compared with standard media like EMB, MSA and NA. Growth
 57

on plant based agar media was observed high in PBM7 when it is compared with standard media
in terms of Cfu/ml. But in other microorganisms i.e. S. aureus and Streptococcus spp. found to
be comparable as standard media like MSA and NA.

All these media were also tested for the growth of environmental contaminants but none of the
media showed any contamination as the petri plates which were kept as control in the safety
cabinet and incubated along with the seeded plates did not exhibit any colony or growth (Bharti
et.al; 2013).

It can be concluded from the present study that on PBM7 plant-based media are found to be
supporting the growth of E. coli, S. aureus and Streptococcus spp. (environmental isolate). Thus,
these plant-based media are cost effective, eco-friendly, easily available and simple to prepare as
compared to currently available synthetic media.
 58

CHAPTER-5
SUMMARY
 61

CHAPTER-6 REFERENCES
Abdullahi II, Abdullahi N, Abdu AM, Ibrahim AS. Proximate, mineral and vitamin analysis of
fresh and canned tomato. Biosciences Biotechnology Research Asia. 2016 Jun 25;13(2):1163-9.
Aigbogun EI, Mohammed SS, Afangide CS, Luka FE, Kangla A. Tomato Juice Agar: An
alternative media for the cultivation of some Aspergillus species. Int J Curr Microbiol Appl Sci.
2018;7(5):788-93.
Alford J, Duguid N (January 1, 2003). Seductions of Rice. Artisan. p. 31. ISBN 978-1-57965-
234-0.
Alves, Loreto, S.H., Linares, E.S., Silveira, C.E., Scheid, C.P., Pereira, L.A., D.I.B. & Santurio,
J.M. Comparison among tomato juice agar with other three media for differentiation of Candida
dubliniensis from Candida albicans. Rev. Inst. Med. trop. S. Paulo. 2006; 48 (3):119-121.

Badole SL, Bodhankar SL. Glycine max (soybean) treatment for diabetes. Bioact. Food Diet.
Interv. Diabetes. 2012 Oct 22; 77:77-82.
Basu S, Bose C, Ojha N, Das N, Das J, Pal M, Khurana S. Evolution of bacterial and fungal
growth media. Bioinformation. 2015;11(4):182.
Bharti NM, Patil S, Kumar A, Sharma PC. Novel Preparations of Culture Media from Plant
Materials for Cultivation of Candida albicans.
Boligon AA, Athayde ML. Importance of HPLC in analysis of plants extracts. Austin
Chromatogr. 2014;1(3):2.
Bonnet M, Lagier JC, Raoult D, Khelaifia S. Bacterial culture through selective and non-
selective conditions: the evolution of culture media in clinical microbiology. New microbes and
new infections. 2020 Mar 1; 34:100622.
Bruck de Souza L, Leitão Gindri A, de Andrade Fortes T, Felli Kubiça T, Enderle J, Roehrs R,
Moura e Silva S, Manfredini V, Gasparotto Denardin EL. Phytochemical analysis, antioxidant
activity, antimicrobial activity, and cytotoxicity of Chaptalia nutans leaves. Advances in
Pharmacological and Pharmaceutical Sciences. 2020 May 1;202
Challis GL, Hopwood DA. Synergy and contingency as driving forces for the evolution of
multiple secondary metabolite production by Streptomyces species. Proceedings of the National
Academy of Sciences. 2003 Nov 25;100(suppl_2):14555-61.
Crisosto H, Ferguson L, Bremer V, Stover E, Colelli G. Fig (Ficus carica L.). InPostharvest
biology and technology of tropical and subtropical fruits 2011 Jan 1 (pp. 134-160e). Woodhead
Publishing.
Diba K, Kordbacheh P, Mirhendi SH, Rezaie S, Mahmoudi M. Identification of Aspergillus
species using morphological characteristics. Pakistan journal of medical sciences. 2007 Oct
1;23(6):867.
 62

Elsawey H, Patz S, Nemr RA, Sarhan MS, Hamza MA, Youssef HH, Abdelfadeel MR, Daanaa
HS, El-Tahan M, Abbas M, Fayez M. Plant broth-(Not bovine-) based culture media provide the
most compatible vegan nutrition for in vitro culturing and in situ probing of plant microbiota.
Diversity. 2020 Nov 4;12(11):418.
Erkmen O. Laboratory Practices in Microbiology. Academic Press; 2021 Feb 6.
Fernando WD. Plants: An international scientific open access journal to publish all facets of
plants, their functions and interactions with the environment and other living organisms. Plants.
2012 Sep;1(1):1.
Fish DN. Optimal antimicrobial therapy for sepsis. American journal of health-system pharmacy.
2002 Feb 1;59(suppl_1): S13-9.
Gautam, A.K., Bhatia, M.K. and Bhadauria, R. Diversity and usage custom of plants of South
Western Himachal Pradesh, India- part 1. J. phyt. 2011; 3(2):24-36, 2011.

Ito S. Marketing of value-added rice products in Japan: germinated brown rice and rice bread.
InProceeding of FAO Rice Conference, 2004 2004.
Jadhav P, Sonne M, Kadam A, Patil S, Dahigaonkar K, Oberoi JK. Formulation of cost effective
alternative bacterial culture media using fruit and vegetables waste. International Journal of
Current Research and Review. 2018 Jan;10(2):6.
Kala CP, Dhyani PP, Sajwan BS. Developing the medicinal plants sector in northern India:
challenges and opportunities. Journal of Ethnobiology and Ethnomedicine. 2006 Dec;2(1):1-5.
Khan ZU, Ahmad S, Mokaddas E, Chandy R. Tobacco agar, a new medium for differentiating
Candida dubliniensis from Candida albicans. Journal of clinical microbiology. 2004
Oct;42(10):4796-8.
Laha GS, Singh R, Ladhalakshmi D, Sunder S, Prasad MS, Dagar CS, Babu VR. Importance and
management of rice diseases: a global perspective. InRice production worldwide 2017 (pp. 303-
360). Springer, Cham.
Mariya A, Haruna AH, Babangida AZ. Phytochemical analysis of some selected indigenous
fruits collected from lokogoma-abuja, nigeria. Journal of Diseases and Medicinal Plants. 2020
Jul 28;6(2):50-5.
Mathew BB, Jatawa SK, Tiwari A. Phytochemical analysis of Citrus limonum pulp and peel. Int
J Pharm Pharm Sci. 2012;4(2):369-71.
Modgil R, Tanwar B, Goyal A, Kumar V. Soybean (Glycine max). InOilseeds: Health Attributes
and Food Applications 2021 (pp. 1-46). Springer, Singapore.
Mohane NR, Tumane PM, Wasnik DD. FORMULATION OF COST EFFECTIVE
ALTERNATIVE MICROBIAL CULTURE MEDIA USING FRUITS AND VEGETABLES
WASTE: A NOVEL APPROACH. Journal of Advanced Scientific Research. 2020 Sep 2;11.
 63

Morton JF. Fruits of warm climates. JF Morton; 1987

Nandhakumar B, Menon T, Kumar CG. A new henna-based medium for the differentiation of
Cryptococcus neoformans. Journal of medical microbiology. 2007 Apr 1;56(4):568-.
Nealson KH. Post-Viking microbiology: new approaches, new data, new insights. Origins of Life
and Evolution of the Biosphere. 1999 Jan;29(1):73-93.
Pant N, Sharma PC, Jatana M, Amit K, Patil S and Sunity Singh, A novel modification of culture
media for cultivation of Cryptococcus neoformans by using different extracts of different plants
from Solan area of Himachal Pradesh (India), Elixir Bio Tech., 45, 2012, 7876-7880.
Ploetz RC. Fusarium wilt of banana. Phytopathology. 2015 Dec 22;105(12):1512-21.
Pommerville JC (2014). Fundamentals of Microbiology (10th ed.). Boston: Jones and
Bartlett. ISBN 978-1-284-03968-9.
Pownall, T.L., Udenigwe, C.C., Aluko, R.E. Amino acid composition and antioxidant properties
of pea seed (um sativum L.) enzymatic protein hydrolysate fractions. J of agricul and food chem.
2010; 58(8):4712–4718.

Saiman L. Microbiology of early CF lung disease. Paediatric respiratory reviews. 2004 Jan
1;5:S367-9.
Sanchez-Monge, R., Lopez-Torrejon, G., Pascual, C.Y., Varela, J., Martin-Esteban, M., Salcedo,
G. Vicilin and convicilin are potential major allergens from pea. Clin & Exp Allergy. 2004;
34(11):1747–1753.

Schwarz S, Enne VI, van Duijkeren E (October 2016). "40 years of veterinary papers in JAC –
what have we learnt?". The Journal of Antimicrobial Chemotherapy. 71 (10): 2681–
90. doi:10.1093/jac/dkw363. PMID 27660260
Shareef SA. Formulation of alternative culture media from natural plant protein sources for
cultivation of different bacteria and fungi. Zanco Journal of Pure and Applied Sciences. 2019
Sep 10;31(4):61-9.
Shoaib M, Muzammil I, Hammad M, Bhutta ZA, Yaseen I. A mini-review on commonly used
biochemical tests for identification of bacteria. A Mini-Review on Commonly used Biochemical
Tests for Identification of Bacteria. 2020 Jun 14;54(1):8-.
Sofowora A. Medicinal Plants and Traditional Medicine in West Arica, John Wily and Sons.
New York. 1982;256.
Thomson RB, Bertram H. Laboratory diagnosis of central nervous system infections. Infectious
Disease Clinics. 2001 Dec 1;15(4):1047-71.
Ugah UI, Nwoba ST. Microbial Media Formulation Using Rice Husks, Yam and Cassava Peels
on Some Selected Microorganisms. Scientific Research. 2018;26(1):122-31.