Shweta ITS& RC Project Report
Transcript of Shweta ITS& RC Project Report
DECLARATION
I hereby declare that the work which has been presented in the
dissertation entitled “To check the activity of α-amylase enzyme,
produce by Bacillus cereus isolated from soil sample collected
from different region of Agra”.
Submitted for the partial fulfilment of the B.E. Biotech is an
authentic record my work carried out under the supervision of
-----------------------.
The matter embodied in this dissertation submitted by me has not
been submitted for a degree of my any other academic in any
other university or examination body in India & abroad.
Place: Agra
SHWETA DASS
Date:
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ACKNOWLEDGEMENT
Commencing with the name of almighty, the most beneficent,
most merciful who do we worship and thine aid we seek.
I am highly grateful and feel my proud privilege to take this
opportunity to express my deepest and heartful sense of
gratitude to, Miss. Deepti Tiwari, Director, ITS & RC, Agra for
his keen interest, affectionate behavior, continued forbearance,
valuable guidance, constructive criticisms and suggestions
without his stimulating guidance tremendous encouragement it
would have not been possible to carry out the present work.
I am also grateful to Mrs. Rashmi Sharma (H.O.D.), Mrs.
Anuradha Chauhan, Miss. Shilpi Gupta, Mr. Arvindra
Kumar Jadaun, for their valuable suggestions different aspects
of the present research work.
I take this opportunity to express my hearty grateful to Dr.
Sanjeev Kumar Sharma, Director, I.E.T. Khandari Campus,
Agra for providing requisite facilities for the study.
It seems quite formal to thank my father Shri Raghuvar Dayal
and mother Smt. Renu Devi, what is mine is there and what I
will be in the near future is certainly will because of them.
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I heartily feel deep regards to my dear madam Miss. Garima
Sharma, who gave me inspiration, affection and always prays for
my better future.
I am also immensely thankful to my elder brother Hardeep Singh
& Harendra Kumar Singh, and sister Pinki kumari for their
affection, bondless co-operation and inspiration.
I am fortunate to have friends like real gem; I am very much
grateful to Pooja & Anjali for their valuable help during the ups
and downs of the life.
Many of my colleagues helped me both morally and academically
at various stages during the period of my study. For this I wish to
record my gratitude and heartiest thanks to Kalpana, Gaurav,
Manisha, And other colleagues but the number is too great to
name them all the number is too great to name them all.
At last but not least, I express my deep sense of gratitude to my
friend’s Ved, Anu, Archarna, Amita, for their affection &
encouragement during the course of my study.
(Shweta Dass)
B.E.
biotechnology
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A
B B
R I
V A
T I
O N
B . cereus Bacillus Cereus
gm gram
Mg milli gram
µl micro litre
ml milli litre
rpm revolution per
minute
d/w distilled water
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TABLE OF CONTENTS
S. NO. TITLE PAGE NO.
1 ABBRIVATION 5
2 AIM OF STUDY 6
3 INTRODUCTION 7-11
4 REVIEW OF LITERATURE 12-44
5 METHOD & MATERIALS 45-69
6 RESULTS 66-69
7 DISCUSSION & CONCLUSION 70-77
8 REFFERENCE 78-88
UV LIGHT ultra violet light
L̊C degree centrigrate
EDTA Ethylene diamine
tetra acetic acid
TAE BUFFER tris acetic acid
EDTA buffer
TE BUFFER tris EDTA buffer
DNA Dioxy ribonucleic
acid
Tm melting
temperature
AIM OF STUDY
On considering the role of B-cereus in several diseases is selected
the study “To check the activity of α-amylase enzyme production
by Bacillus cereus isolated from soil sample collection from
different region of AGRA” with following objectives.
Isolation of B.cereus from different soil sample.
Characterization of isolated B.cereus at Biochemical level.
Characterization of isolated B.cereus at Molecular level.
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To check out the antibacterial activity of several plants against
isolated B.cereus.
INTRODUCTION
Bacillus cereus is a normal inhabitant of the soil, but it can be
regularly isolated from foods such as grains and spices. B. cereus
causes two types of food-borne intoxications (as opposed to
infections). One type is characterized by nausea and vomiting and
abdominal cramps and has an incubation period of 1 to 6 hours. It
resembles Staphylococcus aureus food poisoning in its symptoms
and incubation period. This is the "short-incubation" or emetic
form of the disease. The second type is manifested primarily by
abdominal cramps and diarrhea with an incubation period of 8 to
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16 hours. Diarrhea may be a small volume or profuse and watery.
This type is referred to as the "long-incubation" or diarrheal form
of the disease and it resembles food poisoning caused by
Clostridium perfringens. In either type, the illness usually lasts
less than 24 hours after onset.
The short-incubation form is caused by a preformed, heat-stable
emetic toxin, ETE. The mechanism and site of action of this
toxin are unknown, although the small molecule forms ion
channels and holes in membranes. The long-incubation form of
illness is mediated by the heat-labile diarrheagenic enterotoxin
Nhe and/or hemolytic enterotoxin HBL, which cause intestinal
fluid secretion, probably by several mechanisms, including pore
formation and activation of adenylate cyclase enzymes.
Bacillus cereus is a Gram-positive, spore-forming microorganism
capable of causing foodborne disease at present three
enterotoxins, able to cause the diarrheal syndrome, have been
described: hemolysin BL (HBL), nonhemolytic enterotoxin (NHE)
and cytotoxin K. HBL and NHE are three-component proteins,
whereas cytotoxin K is a single protein toxin. Symptoms caused
by the latter toxin are more severe and may even involve
necrosis. In general, the onset of symptoms is within 6 to 24 h
after consumption of the incriminated food.
In microbiology, the term bacillus means any rod-shaped
microbe (and coccus means a spherical microbe). However,
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Bacillus (written with a capital letter and italicized) refers to a
specific genus of bacteria. The family Bacillaceae are all Gram-
positive, rod-shaped bacteria which form endospores, with two
main divisions:
the anaerobic spore-forming bacteria of the genus
Clostridium
the aerobic or facultatively anaerobic spore-forming bacteria
of the genus Bacillus
Characteristically, Bacillus cultures are Gram-positive when
young, but may become Gram-negative as they age. Bacillus
species are aerobic, sporulating, rod-shaped bacteria which are
ubiquitous in nature. Gram-stained cells, 1 µm wide, 5-10 µm
long, arranged singly or in short chains. The organism produces
heat resistant spores and these may germinate if cooling is too
slow (Hocking, A.D. et al. 1997) [1].
Bacillus endospores are resistant to hostile physical and chemical
conditions, but in addition various Bacillus species have a wide
range of physiologic adaptations which enable them to survive or
thrive in harsh environments, ranging from desert sands and hot
springs to Arctic soils and from fresh waters to marine sediments.
Because the spores of many Bacillus species are resistant to heat,
radiation, disinfectants, and desiccation, they are difficult to
eliminate from medical and pharmaceutical materials and are a
frequent cause of contamination. Bacillus species are well known
in the food industry as spoilage organisms. At the start of this
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video, spores can be seen as the bright, refractile objects seen
under phase contrast microscopy. The second part of the video
show green spores differentiated from pink vegetative cells by a
spore staining procedure:
Fig 1: Bacillus Cereus
Only a few genera of bacteria such as Bacillus and Clostridium are
capable of forming endospores. These are dormant form of the
bacterium that allows it to survive sub-optimal environmental
conditions. Spores have a tough outer covering made of keratin
and are highly resistant to heat and chemicals. The keratin also
resists staining, so specialized procedures are necessary to stain
endospores.
Diarrheal poisoning is caused by heat-labile enterotoxins
produced during vegetative growth of B. cereus in the small
intestine whereas the emetic type of food poisoning is caused by
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the small, heat- and acid-stable cyclic dodecadepsipeptide
cereulide [2] [3]. While enterotoxins are comparatively well
characterized at the molecular and the expression level
(Granum, P.E. 2001) [4], far less is known about the emesis
causing toxin. The chemical structure and characteristics of
cereulide have been studied in some detail but the molecular
basis for its synthesis remains unknown. Cereulide causes cellular
damaging effects in animal models [5] is toxic to mitochondria by
acting as a potassium ionophore [6] and it was involved in
fulminant liver failure in a human case [7]. Recently, it has been
reported that cereulide inhibits human natural killer cells and
might therefore have an immunomodulating effect [8].
In general, the incidence of B. cereus food poisoning is
underestimated since B. cereus is not a reportable disease and
reporting procedures vary between countries. There is a tendency
for many more B. cereus food poisoning cases to be reported in
northern countries. In Norway B. cereus was the most common
microbe isolated from food-borne illnesses in 1990 [9] and it was
responsible for 14% of the outbreaks in Finland in which the
causative agent was identified [10]. B. cereus is a major problem in
convenience food and mass catering. Due to heat and acid
resistance of its spores it is not eliminated by pasteurization or
sanitation procedures. Investigation of food-borne outbreaks in
the German Federal Armed Forces showed that B. cereus was by
far the most frequently isolated pathogen in the retained food
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samples. It was responsible for 42% of the outbreaks reported
between 1985 and 2000.
Since B. cereus is a ubiquitous spore former that cannot be totally
avoided, it is necessary to develop rapid methods to discriminate
hazardous strains from non-toxic strains. The utility of polymerase
chain reaction (PCR) based methods is evident by the 1999
guidelines issued by NCCLS [11] encouraging the use of molecular
methods in clinical laboratories performing bacterial identification
assays. Such an assay would also be advantageous for quality
control in the food industry and could improve food safety
substantially. While for enterotoxic B. cereus strains molecular
diagnostic PCRass ays have been described [12] [13] [14] and
commercial immunological assays are available, for emetic
strains such tools are still missing. The presented PCR system
may fill that gap by providing a molecular assay to rapidly detect
emetic toxin producing B. cereus strains.
REVIEW OF LITERATURE
Bacillus cereus is one of around 60 representatives of the widely
varied Bacillus genus. Along with the very similar species B.
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mycoides, B. thuringiensis and B. anthracis, it comprises the so
called Bacillus cereus group. The differences between these four
species are very small. B. cereus is found frequently as a
saprophyte in soil, water, vegetation and air, from where it is
easily transferred to food, either from the original raw material or
during the food processing. It is common in dried foodstuffs,
spices, cereals, meat, eggs, milk and milk products, cooked and
inappropriately kept food. [15][16][17]
Bacillus cereus is a causative agent of gastrointestinal and non-
gastrointestinal diseases. Bacillus cereus causes two distinct food
poisoning syndromes:
Rapid-onset emetic syndrome characterized by nausea
and vomiting. Nausea and vomiting begins one to five hours
after contaminated food is eaten. Boiled rice that is held for
prolonged periods at ambient temperature and then quick-
fried before serving is a frequent cause, although dairy
products or other foods may also be responsible.
Slow-onset diarrhoeal syndrome. Diarrhoea and
abdominal pain occurs 8 to 16 hours after consumption of
contaminated food. This is associated with a variety of foods,
including meat and vegetable dishes, sauces, pastas,
desserts, and dairy products.
Besides its food poisoning potential, B. cereus has been shown to
be responsible for wound and eye infections, as well as systemic
infections [18]. Recently, it has been reported that systemic
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complications of B. cereus infections in premature neonates might
be at least partly related to enterotoxins [19]. However, in general
the role of the diverse toxins and virulence factors of B. cereus in
systemic infections is poorly studied. The development of
molecular tools will be necessary to allow a rapid characterization
of virulence mechanisms of clinical B. cereus isolates.
SCIENTIFIC CLASSIFICATION
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Bergey’s Manual contains six sections that describe all Gram
positive bacteria except the actinomycetes. Most of these
bacteria are distributed among the first sections on the basis of
their general shape (weather they rods or bacilli, cocci or
irregular) and their ability to form endoscope.
In Bergey's Manual of Systematic Bacteriology (1st ed. 1986), the
G+C content of known species of Bacillus ranges from 32 to 69%.
This observation, as well as DNA hybridization tests, revealed the
genetic heterogeneity of the genus.
In Bergey's Manual of Systematic Bacteriology (2nd ed. 2004),
phylogenetic classification schemes landed the two most
prominent types of endospore-forming bacteria, clostridia and
bacilli, in two different Classes of Firmicutes, Clostridia and
Bacilli. Clostridia includes the Order Clostridiales and Family
Clostridiaceae with 11 genera including, Clostridium. Bacilli
include the Order Bacillales and the Family Bacillaceae. In this
family there are 37 new genera on the level with Bacillus.
TAXONOMIC CLASSIFICATION14
Kingdom :- Bacteria
Phylum :- Firmicutes
Class :- Bacilli
Order :- Bacillales
Family :- Bacillaceae
Genus :- Bacillus
Species :- cereus
HISTORY
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In 1887, Bacillus cereus isolated from air in a cowshed by
Frankland and Frankland.
In 1906, B. cereus was first associated with food poisoning in
Europe. Outbreaks of food poisoning caused by aerobic,
sporeforming bacilli termed “anthracoid” or “pseudoanthrax”
were reported.
In 1950, Steinar Hauge in Norway provided the first
complete account of B. cereus poisoning, and proved that
this microorganism is a human pathogen. From 1947–1949,
Hauge investigated four outbreaks of food poisoning with a
total of 600 persons affected. The food vehicle in all four
outbreaks was vanilla sauce prepared from corn starch, rich
in B. cereus spores. Hauge found that the corn starch used in
this case had ~104 spores of B. cereus per gram. The
dessert was prepared and stored at room temperature until
it was served and eaten the next day. All individuals who ate
the dessert had clinical symptoms of food poisoning. To
provide evidence that B. cereus was the cause of food
poisoning, Hauge demonstrated Koch’s postulates by
consuming a culture of the isolated B. cereus strain. He grew
B. cereus to a level of 4×106 cells per ml, and drank 200 ml
of bacterial suspension. After 13 hrs, the symptoms of food
poisoning started.
Since 1950, many outbreaks from a variety of foods
including meat and vegetable soups, cooked meat and
poultry, fish, milk and ice cream were described in Europe.
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In 1954, experiments with volunteers in USA failed to
confirm Hauge’s observations.
In 1969, the first well-characterized B. cereus outbreak in the
USA was documented.
Since 1971, a number of B. cereus poisonings of a different
type, called the vomiting type, were reported. This type of
poisoning was characterized by an acute attack of nausea
and vomiting 1–5 hrs after consumption of the incriminated
meal. Sometimes, the incubation time was as short as 15–30
min or as long as 6–12 hrs. Almost all the vomiting type
outbreaks were associated with consumption of cooked rice.
This type of poisoning resembled staphylococcal food
poisoning.
SOURCES OF BACILLUS CEREUS 17
1.Wide distribution in soil, dust and air
B. cereus is widely distributed in nature and can be found in
soil, dust, air, water and decaying matter. Its ability to form
spores allows survival through all stages of food-processing,
other than retorting.
2.Carried by humans and animals
Human: Humans are not a significant source of food
contamination by B. cereus. This organism already exists on
many foods and can therefore be transiently carried in the
intestine of healthy humans (0-43%).
Animal: Animals can carry B. cereus on parts of their body.
May occasionally cause mastitis in cows.
3.In many food products
Raw foods of plant origin are the major source of B. cereus.
The widespread distribution of the organism, the ability of
spores to survive dried storage and the thermal resistance of
spores, means that most ready-to-eat foods will contain B.
cereus and will require control measures to prevent growth,
especially after cooking has eliminated competing flora.
Strains producing emetic toxin grow well in rice dishes and
other starchy foods, whereas strains producing diarrhoeal
toxin grow in a wide variety of foods from vegetables and
salads to meat and casseroles. Numerous dried herbs and
spices and dehydrated foods have been shown to contain B.
cereus.
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4.Dairy products
Rice and cooked oriental foods
It’s not just rice, this is just the most well known example
of foods that can become contaminated. Other cooked
cereals such as cous and bulghur wheat can also be
affected, as can pasta, potatoes, pastries, any foods with
sauces, such as casseroles and pies. Even salads have been
found to harbor Bacillus cereus spores and actively growing
bacteria.
Spices and spice mixes
Dried products (flour, dry milk, pudding, soup mix)
5.Meats
Microorganisms control in meat products is the major concern
in the preparation of high quality foods (Jo et al., 2004) [20].
The hygienic state of animals prior, during and after slaughter
can be critical to the finished product quality (Satin, 2002) [21]. During slaughtering process the meat is exposed to many
sources of Bacillus cereus contamination (Lawrie, 1998) [22].
The incidence of Bacillus cereus is higher in cooked and
processed (ground beef) meat than in raw meat samples
(Nortje et al., 1999; Mosupye & Von Holg, 2000) [23] [24].
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STRUCTURE OF BACILLUS CEREUS
Like most Gram-positive bacteria the surface of the Bacillus
cereus is complex and is associated with their properties of
adherence, resistance and tactical responses. The vegetative cell
surface is a laminated structure that consists of a capsule, a
proteinaceous surface layer (S-layer), several layers of
peptidoglycan sheeting, and the proteins on the outer surface of
the plasma membrane.
Fig2: Surface of a Bacillus cereus Transmission E.M. C=Capsule;
S=S-layer; P=Peptidoglycan.
Surface layer (S-layer) :-
A regularly ordered protein or glycoprotein layer (S-layer) has
been detected as the outermost component of several gram-
negative and gram-positive organisms (Beveridge T J 1997,
Messner P, Sleytr U B 1992) [25] [26]. The functions of the S-
layer in bacteria are not completely understood. It has been
suggested that the S-layer mediates the adhesion to avian
intestinal epithelial cells in Lactobacillus acidophilus and to
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collagen in Lactobacillus crispatus [27] Increased virulence and
resistance to phagocytosis (Noanao B, Trust T J 1997) [28]
have been associated with the presence of the S-layer in
animal pathogens. Ellar and Lundgren (Ellar D J, Lundgren D
G 1967) [29] described the presence of an S-layer on the
surface of B. cereus .
Capsule :-
Capsule synthesis in Gram positive bacteria falls into two
catagories; production of polyglutamic acid and polysaccharide
capsule. While most laboratory strain of B.subtilis do not
produce significant capsule material, the genome sequence
indicates that they possess the genes required for production
of each type of capsule. (Ueda, S.; 1989) [30].
Cell Wall :
The variability of cell wall structure that is common in many
Gram-positive bacteria does not occur in the genus Bacillus.
The vegetative cell wall of almost all Bacillus species is made
up of a peptidoglycan containing meso-diaminopimelic acid
(DAP). This is the same type of cell wall polymer that is nearly
universal in Gram-negative bacteria, i.e., containing DAP as the
diamino acid in position 3 of the tetrapeptide. In some cases,
DAP is directly cross-linked to D-alanine, same as in the
Enterobacteriaceae; in other cases, two tetrapeptide side
chains of peptidoglycan are spanned by an interpeptide bridge
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between DAP and D-alanine, which is characteristic of most
Gram-positive bacteria.
In addition to peptidoglycan in the cell wall, all Bacillus species
contain large amounts of teichoic acids which are bonded to
muramic acid residues. The types of glycerol teichoic acids
vary greatly between Bacillus species and within species. As in
many other Gram-positive bacteria, lipoteichoic acids are found
associated with the cell membranes of Bacillus species.
The cell wall forms the barrier between the environment and
the bacterial cell. It is also responsible for maintaining the
shape of the cell and withstanding the cell's high internal
turgor pressure (Schaechter 2006) [31].
Fig3: Mechanism of cell wall
The cell wall synthetic enzymes (eg. penicillin binding proteins
and autolysins) are produced intracellularly but their sites of
action are extracellular, i.e. within the cell wall. Therefore cell
wall synthesis requires signaling between the cell wall and the
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cytoplasmic compartments to coordinate the production of
precursors/enzymes with their utilization. (Kevin M. Devine et
al. 2008) [32].
Flagella :-
The flagellum is essential for active movement of individual
cells in a liquid environment (swimming) and for chemotaxis
and plays an important role in interaction with surfaces as a
sensor of medium viscosity (McCarter, L., et.al.; 1988) [33] .
When bacterial flagella are examined by electron microscopy
(Abram, D., A. E. Vatter, and H. Koffler. 1966 ;) [34] they
are found to be composed of three morphologically
distinguishable sections: a long flagellar filament, a hook like
terminal structure, and a basal region which is attached to the
cell membrane.
Swarming can be considered a strategy for rapid spread over
solid surfaces in the environment and for active colonization of
mucosal surfaces in infected hosts (Allison, C, et.al., 1992) [35].
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Fig4: Different type of B.cereus
Fig5: Electron microscopic Structure of
Flagella
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Endospore :-
Endospores were first described by Cohn in Bacillus subtilis
and later by Koch in the pathogen, Bacillus anthracis. Cohn
demonstrated the heat resistance of endospores in B. subtilis,
and Koch described the developmental cycle of spore
formation in B. anthracis. Endospores are so named because
they are formed intacellularly, although they are eventually
released from this mother cell or sporangium as free spores.
Endospores have proven to be the most durable type of cell
found in Nature, and in their cryptobiotic state of dormancy
they can remain viable for extremely long periods of time,
perhaps millions of years.
fig 6- spores of bacillus
Pathogenesis of Bacillus cereus
B. cereus is responsible for a minority of food borne illnesses (2–
5%), causing severe nausea, vomiting and diarrhea (Kotiranta A,
Lounatmaa K, Haapasalo M 2000) [36]. Generally speaking,
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Bacillus foodborne illnesses occur due to survival of the bacterial
endospores when food is improperly cooked. This problem is
compounded when food is then improperly refrigerated, allowing
the endospores to germinate (McKillip JL 2000) [37]. Bacterial
growth results in production of enterotoxins, one of which is
highly resistant to heat and to pH between 2 and 11, ingestion
leads to two types of illness, diarrheal and emetic (vomiting)
syndrome.
The diarrheal type is associated with a wide-range of foods, has
an 8–16.5 hour incubation time and is associated with diarrhea
and gastrointestinal pain. Also known as the long-incubation
form of B. cereus food poisoning, it might be difficult to
differentiate from poisoning caused by Clostridium perfringens.
The emetic form is commonly caused by rice that is not cooked
for a time and temperature sufficient to kill any spores
present, then improperly refrigerated. It can produce a toxin
which is not inactivated by later reheating. This form leads to
nausea and vomiting 1–5 hours after consumption. It can be
difficult to distinguish from other short-term bacterial food
borne pathogens, e.g., Staphylococcus aureus).
If rice is cooked at, or over 100 degrees Celsius for 20 minutes or
more bacillus cereus cannot survive, therefore eliminating
possible food-poisoning. It was previously thought that the timing
of the toxin production might be responsible for the two different
types, but in fact the emetic syndrome is caused by a toxin called
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cereulide that is found only in emetic strains and is not part of the
"standard toolbox" of B. cereus. Cereulide, a dodecadepsipeptide
produced by non-ribosomal peptide synthesis (NRPS), which is
somewhat unusual in itself. Cereulide is believed to activate 5-HT
receptors leading to increased afferent vagal stimulation (Agata
N, Ohta M, Mori M, Isobe M 1995) [38].
Toxins Production
Bacillus cereus produces one emetic toxin (ETE) or Cereulide and
three different enterotoxins: HBL, Nhe, and EntK.
Two of the three enterotoxins are involved in food poisoning. They
both consist of three different protein subunits that act together.
One of these enterotoxins (HBL) is also a hemolysin; the second
enterotoxin (Nhe) is not a hemolysin. The third enterotoxin (EntK)
is a single component protein that has not been shown to be
involved in food poisoning. All three enterotoxins are cytotoxic
and cell membrane active toxins that will make holes or channels
in membranes.
Cereulide is a small, heat and acid stable cyclic
dodecadepsipeptide which is chemically closely related to the
potassium ionophore valinomycin (Agata N, Mori M, Ohta M,
Suwan S, Ohtani I, Isobe M 1994) [39]. It is toxic to
mitochondria by acting as a potassium ionophore and has been
reported to inhibit human natural killer cells [40]. According to its
chemical structure it has been shown that this toxin is produced
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by a nonribosomal peptide synthetase (NRPS), but its exact
genetic organization and biochemical synthesis is unknown.
The non-hemolytic enterotoxin (Nhe) is one of the three-
component enterotoxins responsible for diarrhea in Bacillus
cereus food poisoning. Nhe is composed of NheA, NheB and NheC.
The three genes encoding the Nhe components constitute an
operon. The nhe genes have been cloned separately, and
expressed in either Bacillus subtilis or Escherichia coli. Separate
expression showed that all three components are required for
biological activity.
The hemolytic enterotoxin, HBL, is encoded by the hblCDA
operon. The three protein components, L1, L2 and B, constitute a
hemolysin. B is for binding; L1 and L2 are lytic components. This
toxin also has dermonecrotic and vascular permeability activities,
and it causes fluid accumulation in rabbit ileal loops.
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APPLICATIONS OF B. CEREUS
Symbiosis
B. cereus competes with other microorganisms such as
Salmonella and Campylobacter in the gut, so its presence reduces
the numbers of those microorganisms. In food animals such as
chickens [41], rabbits, and pigs, some harmless strains of B. cereus
are used as a probiotic feed additive to reduce Salmonella in the
intestines and cecum. This improves the animals' growth as well
as food safety for humans who eat their meat.
Antibiotic Production
Bacillus antibiotics share a full range of antimicrobial activity:
bacitracin, pumulin, laterosporin, gramicidin and tyrocidin are
effective against Gram-positive bacteria; colistin and polymyxin
are anti-Gram-negative; difficidin is broad spectrum; and
mycobacillin and zwittermicin are anti-fungal.
As in the case of the actinomycetes, antibiotic production in the
bacilli is accompanied by cessation of vegetative growth and
spore formation. This has led to the idea that the ecological role
of antibiotics may not rest with competition between species, but
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with the regulation of sporulation and/or the maintenance of
dormancy.
Antibiotics produced by the aerobic sporeformers are often, but
not always, polypeptides. Known antibiotic producers are Bacillus
cereus (e.g. cerexin, zwittermicin), Bacillus circulans (e.g.
circulin), Brevibacillus laterosporus (e.g. laterosporin), Bacillus
licheniformis (e.g. bacitracin), Paenibacillus polymyxa (e.g.
polymyxin, colistin), Bacillus pumilus (e.g. pumulin) and Bacillus
subtilis (e.g. polymyxin, difficidin, subtilin, mycobacillin).
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MEDICINAL PLANTS
The use of medicinal plants as a source for relief from illness can
be traced back over five millennia to written documents of the
early civilization in China, India and the Near east, but it is
doubtless an art as old as mankind. Neanderthals living 60,000
years ago in present day Iraq used plants such as hollyback,
these plants are still widely used in ethnomedicine around the
world .[42]
1. PEEPAL
Scientific Classification :
Kingdom :- Plantae
Division :- Magnoliophyta
Class :- Magnoliopsida
Oder :- Rosales
Family :- Moraceae
Genus :- Ficus
Species :- F.religiosa
Scientific Name :- Ficus religiosa
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Other names: Bargad, Bor, Ber, Ala and Pedda mari,
Navagrodha, Ala mara, Bar, Vad, Vatnam, Bahupada, Peddamarri,
Al are the other names used for the Banyan tree. Indians call it a
wish fulfilling tree.
1. Description: Banyan tree is a huge tree with very extensive
branches. It is said that at one time more than 10,000
people can sit under its shade at one time. It is evergreen
tree.
2. Medicinal uses: The Banyan tree also has several
medicinal properties. Its leaf, bark, seeds and fig are used
for the variety of disorders like diarrhea, polyuria, dental,
diabetes and urine disord. The wood of the Banyan tree is
used in making door panels, boxes and the other items.
3. Other uses: In India its edible leaves are used as the plates.
It is planted for the soil conservation. Wood is used for well
curbs, door panels, boxes, furniture etc. It is suitable for
paper pulp. The wood of the aerial roots is stronger and is
used for the tent polesand card yokes.
4. Cultural importance: Banyan tree is respected and is
considered as sacred by the people in India. In the sacred
Hindu Book “Bhagwat Gita” Lord Krishna has sung praises on
the Banyan tree. People in India grow Banyan tree closer to
the Peepal tree. As Banyan tree is considered as the male
plant closely related to the Peepal tree.
32
2.GUAVA
Scientific classification:
1. Guavas are plants in the myrtle family (Myrtaceae) genus
Psidium (meaning "pomegranate" in Latin),[43] which contains
about 100 species of tropical shrubs and small trees. They are
native to Mexico, Central America, and northern South America.
33
Kingdom Plantae
Division :- Angiosperms
Class :- Eudicots
Subclass :- Rosids
Oder :- Myrtales
Family :- Myrtaceae
Subfamily :- Myrtoideae
Genus :- Psidium
Species :- P.guajava
Scientific Name :- Psidium guajava
2. Uses:-The fruit are not only relished by humans, but by many
mammals and birds as well. The spread of introduced guavas
owes much to this fact, as animals will eat the fruit and disperse
the seeds in their droppings.
In several tropical regions, including Hawaii, some species
(namely Strawberry Guava, P. littorale, and to a lesser extent
Apple Guava Psidium guajava) have become invasive species. On
the other hand, several species have become very rare due to
habitat destruction and at least one (Jamaican Guava, P.
dumetorum), is already extinct.
3. Guava fruit:-Guava fruit, usually 4 to 12 cm long, are round or
oval depending on the species. The outer skin may be rough,
often with a bitter taste, or soft and sweet. Varying between
species, the skin can be any thickness, is usually green before
maturity, but becomes yellow, maroon, or green when ripe.
4. Culinary uses:-In Hawaii, guava fruit is eaten with soy sauce
and vinegar. Occasionally, a pinch of sugar and black pepper are
added to the soy sauce and vinegar mixture. The guava fruit is
cut up and dipped into the sauce.
5. Nutritional value:-Guavas are often included among
superfruits, being rich in dietary fiber, vitamins A and C, folic acid,
and the dietary minerals, potassium, copper and manganese.
Having a generally broad, low-calorie profile of essential nutrients,
34
a single common guava (P. guajava) fruit contains about four
times the amount of vitamin C as an orange. [44]
3.TULSI PLANT
Scientific classification:
1. Fever & Common
Cold:-The leaves of
basil are specific for
many fevers. During
the rainy season, when
malaria and dengue
fever are widely
prevalent, tender
leaves, boiled with tea,
act as preventive against theses diseases. In case of acute fevers,
a decoction of the leaves boiled with powdered cardamom in half
a liter of water and mixed with sugar and milk brings down the
temperature. The juice of tulsi leaves can be used to bring down
35
Kingdom Plantae
Division :- Magnoliophyta
Class :- Magnoliopsida
Order :- Lamiales
Family :- Lamiaceae
Genus :- Ocimum
Species :- O. tenuiflorum
Scientific name :- Ocimum Sanctum
fever. Extract of tulsi leaves in fresh water should be given every
2 to 3 hours. In between one can keep giving sips of cold water. In
children, it is every effective in bringing down the temperature.
2. Coughs:-Tulsi is an important constituent of many Ayurvedic
cough syrups and expectorants. It helps to mobilize mucus in
bronchitis and asthma. Chewing tulsi leaves relieves cold and flu.
3. Skin Disorders:-Applied locally, basil juice is beneficial in the
treatment of ringworm and other skin diseases. It has also been
tried successfully by some naturopaths in the treatment of
leucoderma.
4. Teeth Disorder:- The herb is useful in teeth disorders. Its
leaves, dried in the sun and powdered, can be used for brushing
teeth. It can also be mixed with mustered oil to make a paste and
used as toothpaste. This is very good for maintaining dental
health, counteracting bad breath and for massaging the gums. It
is also useful in pyorrhea and other teeth disorders.
5. Eye Disorders:-Basil juice is an effective remedy for sore eyes
and night-blindness, which is generally caused by deficiency of
vitamin A. Two drops of black basil juice are put into the eyes
daily at bedtime.
36
4.NEEM TREE
Scientific classification:
37
1. Fruit:-The fruit is a
smooth (glabrous) olive-
like drupe which varies in
shape from elongate oval
to nearly roundish, and
when ripe are 1.4-2.8 x
1.0-1.5 cm. The fruit skin
(exocarp) is thin and the
bitter-sweet pulp (mesocarp) is yellowish-white and very fibrous.
The mesocarp is 0.3-0.5 cm thick. The white, hard inner shell
(endocarp) of the fruit encloses one, rarely two or three,
elongated seeds (kernels) having a brown seed coat.
2. Uses:-Neem products are also used in selectively controlling
pests in plants. It is considered a major component in Ayurvedic
and Unani medicine and is particularly prescribed for skin disease.[45]
All parts of the tree are said to have medicinal properties
(seeds, leaves, flowers and bark) and are used for preparing
many different medical preparations.
Part of the Neem tree can be used as a spermicide [46]
Neem oil is used for preparing cosmetics (soap, neem
shampoo - Sunsan herbal, balms and creams, for example
Margo soap), and is useful for skin care such as acne
38
Kingdom Planate
Division :- Magnoliophyta
Class :- Magnoliopsida
Order :- Sapindales
Family :- Meliaceae
Genus :- Azadirachta
Species :- A. Indica
Scientific name :- Azadirachta
indica
treatment, and keeping skin elasticity. Neem oil has been
found to be an effective mosquito repellent.
Practitioners of traditional Indian medicine recommend that
patients suffering from chicken pox sleep on neem leaves.
Aqueous extracts of neem leaves have demonstrated
significant antidiabetic potential.
3. Neem is a fast-growing tree that can reach a height of 15–20
m (about 50–65 feet), rarely to 35–40 m (115–131 feet). It is
evergreen, but in severe drought it may shed most or nearly all
of its leaves
4.Leaves:-The opposite, pinnate leaves are 20–40 cm (8 to 16
in.) long, with 20 to 31 medium to dark green leaflets about 3–
8 cm (1 to 3 in.) long. The terminal leaflet is often missing. The
petioles are short.
5. Flowers:-The (white and fragrant) flowers are arranged
axillary, normally in more-or-less drooping panicles which are
up to 25 cm (10 in.) long. The inflorescences, which branch up
to the third degree, bear from 150 to 250 flowers. An individual
flower is 5–6 mm long and 8–11 mm wide. Protandrous,
bisexual flowers and male flowers exist on the same individual.
5. BANYAN TREE
39
Scientific classification:
Kingdom Plantae
Division :- Magnoliophyta
Class :- Magnoliopsida
Order :- Urticales
Family :- Moraceae
Genus :- Ficus
Scientific
Name
:- Ficus Benghalensis
1. A banyan is a fig that starts its life as an epiphyte when its
seeds germinate in the cracks and crevices on a host tree (or on
structures like buildings and bridges). "Banyan" often refers
specifically to the Indian Banyan or Ficus benghalensis, the
National tree of India,[47] though the term has been generalized to
include all figs that share a unique life cycle, and systematically
to refer to the subgenus Urostigma.[48]
2. Classification:-
Ficus microcarpa , which is native from Sri Lanka through
New Caledonia and is a significant invasive species
elsewhere.
40
The Central American Banyan (Ficus pertusa) is native to
Central America and northern South America, from southern
Mexico south to Paraguay.
The Shortleaf Fig (Ficus citrifolia) is native to southern
Florida, the Caribbean Islands, Central America and South
America south to Paraguay. One theory is that the
Portuguese name for F. citrofolia, "Os Barbados", gave
Barbados its name.
3. Other:- Banyan tree is the National Tree of India.
The banyan is part of the coat of arms of Indonesia. It is
meant to symbolize the unity of Indonesia - one country with
many far-flung roots. As a giant tree, it also symbolizes
power. Soeharto used it as a logo for his party, the Golongan
Karya (Golkar), taking advantage of the deeply rooted belief
of his fellow-countrymen and women in the sacred nature of
the banyan
Royal Navy and Royal Australian Navy personnel use the
term "banyan" to mean a spell ashore for a barbecue on a
deserted beach. "Banyan Rig" denotes the casual (and often
traditionally tasteless) clothes worn for these events.
The underground roots of a banyan species found in the
Amazon are cut into 10 cm lengths, dried and smoked
regularly to relieve pain. This practice originated in the
Amazon. There are no visible side effects
41
4. Location:- The largest such tree is now found in Kolkata in
India. One of the most famous of banyan trees was planted on
the island of Kabirvad in Gujarat. The City of Vadodara &
Valsad in western India are named after the Banyan Tree.
Ta Prohm in the Angkor Wat temple complex of Cambodia is
well known for the giant banyans that grow up, around and
through its walls.
Several banyans can be found near downtown Hilo, Hawaii.
Some of them were planted by celebrities throughout the
20th century and form the Banyan Drive.
5. Culture:- Also in Hindu culture, the banyan tree is also
called kalpavriksha meaning 'wish fulfilling divine tree'. In
modern parlance in the Hindi language, it is known as Bargad,
Vatavriksh, and Barh.
Buddha is believed to have achieved enlightenment in
Bodhgaya in India while meditating under a banyan tree of
the species Sacred Fig. The tree is known as Bodhi Tree
In Buddhism's Pali canon, the banyan (Pali: nigrodha) is
referenced numerous times.[49] Typical metaphors allude to
the banyan's epiphytic nature, likening the banyan's
supplanting of a host tree as comparable to the way sensual
desire (kāma) overcomes humans.[50]
42
6. SARACA ASOCA
Scientific classification:
1. The Ashoka is a rain-forest
tree. Its original distribution
was in the central areas of
the Deccan plateau, as well
as the middle section of the
Western Ghats in the
western coastal zone of the Indian Subcontinent.
2. The ashoka tree is closely associated with the Yakshi
mythological beings
3. As an artistic element, often the tree and the Yakshi are subject
to heavy stylization. Some authors hold that the young girl at the
foot of this tree is based on an ancient fertility symbol. [51]
43
Kingdom Plantae
Division :- Angiosperms
Class :- Eudicots
Order :- Fabales
Family :- Fabaceae
Genus :- Saraca
Species :- S. asoca
Scientific
name
:- Saraca asoca
4. Then the tree magically bent down for her and she grasped a
branch. At that moment the Buddha emerged from her right
side.[52]
5. This tree has a multitude of names in Indian literature. Some
names for the Ashoka tree and its flowers include:
7. POMEGRANATE
Scientific classification:
1. A pomegranate (Punica granatum) is a fruit-bearing
deciduous shrub or small tree growing between five and eight
meters tall. The pomegranate is mostly native to the Iranian
Plateau and the Himalayas in north Pakistan and Northern India.
Introduced into Latin America and California by Spanish settlers in
44
Kingdom Plantae
Division :- Magnoliophyta
Class :- Magnoliopsida
Subclass :- Rosidae
Order :- Myrtales
Family :- Lythraceae
Genus :- Punica
Speces :- P. Granatum
Scentific Name :- Punica
granatum
1769, pomegranate is now cultivated in parts of California and
Arizona for juice production.[53]
2. Description:- The leaves are opposite or sub-opposite, glossy,
narrow oblong, entire, 3–7 cm long and 2 cm broad. The flowers
are bright red, 3 cm in diameter, with four to five petals (often
more on cultivated plants). Some fruitless varieties are grown for
the flowers alone. The edible fruit is a berry and is between a
lemon and a grapefruit in size, 5–12 cm in diameter with a
rounded hexagonal shape, and has thick reddish skin and around
600 seeds.[54]
3. Uses:- The entire seed is consumed raw, though the watery,
tasty aril is the desired part. The taste differs depending on the
subspecies of pomegranate and its ripeness. The pomegranate
juice can be very sweet or sour, but most fruits are moderate in
taste, with sour notes from the acidic tannins contained in the aril
juice.
4. Ayurvedic medicine:-In the Indian subcontinent's ancient
Ayurveda system of medicine, the pomegranate has extensively
been used as a source of traditional remedies for thousands of
years.[55]
The rind of the fruit and the bark of the pomegranate tree is used
as a traditional remedy against diarrhea, dysentery and intestinal
parasites. [55]
45
5. Health Benefits:- In preliminary laboratory research and
clinical trials, juice of the pomegranate may be effective in
reducing heart disease risk factors, including LDL oxidation,
macrophage oxidative status, and foam cell formation. [56][57][58]
In a limited study of hypertensive patients, consumption of
pomegranate juice for two weeks was shown to reduce systolic
blood pressure by inhibiting serum angiotensin-converting
enzyme.[59] Juice consumption may also inhibit viral infections [60]
while pomegranate extracts have antibacterial effects against
dental plaque.[61] [62]
MATERIAL & METHODS
REQUIREMENT
Conical flask
15 Vile
Pipette
Water bath
Centrifuge
Electronics analytical
balance
Autoclave
Agarose gel
electrophoresis assembly
Casting tray
Comb
Balancer
Deep freezer
PCR( Thermal cycle)
Beakers
Aluminium foil
Oven
Incubator loop
Cotton
Matching box
WASHING
46
Firstly we discard the Petri dish. In which Petri dishes are wrap
with Paper and Aluminum foil. And tapping with tap on to the
wrapped Petri dish.
Then placed it in to the Autoclave.
Set the Autoclave at 121 LC for 15 min. The temperature was 15
psi.
Now we use the detergent for washing the Petri dishes.
To dry the Petri dish we use the Hot air oven at 80 LC for 30
min. Before drying we wrap the Petri dish by Paper.
Store the wrapped Petri dishes for further use.
We use the detergent for washing the Tip.
To dry the Tip we use the Hot air oven at 37 LC for 30 min.
Before drying, place all the tips in to the tip box. Then we wrap
the tip box by Paper.
Store the wrapped Tip box for further use.
STERILIZATION
GLASSWARE:
To take the glassware like Petri dishes, conical flasks, Jars, Test
tubes, etc.
Wrap the glassware by Paper and Aluminum foil. And tapping
by tap on to the wrapped glassware.
Take some water in to the Autoclave and place the wrap
glassware.
Set the Autoclave at 121 LC for 15 min. And Pressure was 15 Psi.
Store the wrapped glassware for further use.
47
PLASTIC WARE:
To take the plastic ware like tips of pipette, Eppendrofs or vial,
etc.
All tips are place in to the tip box and vile are in to the vile box.
Wrap the boxes by Paper and Aluminum foil. And tapping by
tap on to the wrapped box.
Take some water in to the Autoclave and place the wrap
glassware.
Set the Autoclave at 121 LC for 15 min. And Pressure was 15 Psi.
Store the wrapped boxes for further use.
Sterilize the platinum loop by the Flame (direct heat).Whenever
the loop was red hot.
CHEMICAL STERILIZATION:
Before doing practical we wash our hand by Alcohol.
Wipe the surface area of performing experiment by the Alcohol.
Some time we wiped the glassware like Petri dish, Slide, etc.
with alcohol also.
48
SAMPLE COLLECTION:-
5 samples were collected from different region of Agra.
S.No
.
Area of
Collection
Type of
Sample
Type of
Sample
1. Shastripuram , Agra Soil R 1
2. Runkata , Agra Soil R 2
3. Keetham ,Agra Soil R 3
4. Rambagh , Agra Soil R 4
5. Sikandra ,Agra Soil R 5
49
SAMPLE PREPARATION:
Weigh 1gm of soil from different sample and suspended it
individually in 10 ml. distilled water containing test tubes. Mix the
samples properly and heat it at 80°C in hot air oven for one hour.
This step allows the killing of all vegetative cells present in the
sample, only spores will remain.
CULTURING:
Bacillus cereus was isolated from the above sample by streaking
the sample on Nutrient agar Medium which is Basal media for all
microorganisms.
PREPARATION OF NUTRIENT AGAR MEDIA:
Ingredients gm/liter
Peptic digest of animal
tissue
:- 5.00
Beef extract :- 1.50
Yeast extract :- 1.50
Sodium chloride :- 5.00
Agar :- 15.00
50
Final pH (at 25°C) :- 7.4 ± 0.2
PROCEDURE-
All the ingredients were suspended in desired amount in the flask
containing distilled water, stirred well to dissolve. Heat to boiling
to dissolve the medium completely. The pH was adjusted to 7.4 ±
0.2 by adding 10N Sodium hydroxide. This medium was
dispensed into culture flasks, autoclaved at 121oC at 15 lb
pressure for 15 min and then allowed to cool at room temperature
and poured in petridish. After solidification the medium was
streaked with samples collected.
The colonies which appeared abundant, forming opaque, creamy
on agar (pH 7.0) were further grown on Bacillus differential media.
This media is used to differentiate Bacillus subtilis and Bacillus
cereus based on their capability to ferment Mannitol.
PREPARATION OF BACILLUS DIFFERENTIAL
MEDIA
Ingredients gm/liter
Yeast autolysate :- 0.20
Mannitol :- 5.00
Phosphate :- 1.00
Potassium :- 0.20
Magnesium :- 0.20
Bromo cresol purple :- 0.0075
51
Agar :- 15.40
Final pH (at 25°C) :- 7±2
PROCEDURE-
All the ingredients were taken in the flask, stirred well to
dissolve.
The pH was adjusted to 7.4±0.2 by adding NaCl or HCl.
This medium was dispensed into culture flasks, autoclaved at 121oC at 15 lb pressure for 15 min.
Then allowed to cool at room temperature and poured in petridish.
After solidification the medium was streaked with samples collected
The colonies which appeared white on Bacillus differentiation agar
were collected and preserve as pure culture in nutrient broth.
These pure cultures were further assayed by biochemical test and
Gram staining.
IDENTIFICATION
1.GRAM’S STAINING:
Reagents-
Gram’s
stain
:- Crystal Violet
52
Moderant
:- Grams Iodine
Decolorizing
agent
:- 70% Alcohol
Counter stain :- Safranin
Procedure:-
The smear was prepared on sterilized glass slide.
The smear was fixed by passing over the flame.
The smear was flooded with crystal violet and incubated for
2 min.
The smear was washed with tap water.
The smear was flooded with gram’s iodine for 2 min.
The smear was washed with tap water.
The smear was decolorized with 70% alcohol for 30 sec.
The smear was washed with tap water.
The smear was counter stained with safranin for 2 min.
The smear was washed with tap water, air dried and
observed under oil immersion microscope.
2.ENDOSPORE STAINING:
Reagent
53
Gram’s stain Crystal Violet
Counter stain Safranin
Procedure-
Place a strip of blotting paper over the slide.
Place the covered slide over a screened water bath and then
saturate blotting paper with primary stain malachite green.
Allow the slide to sit over the steaming water bath for 5
minutes, reapplying stain if it begins to dry out.
Remove blotting paper and rinse slide with water until water
runs clear.
Flood slide with the counterstain safranin for 20 seconds and
then rinse.
View specimen under oil immersion lens with light
microscope.
BIOCHEMICAL TEST
54
1.CATALASE TEST:
Catalase test is used to detect the presence of the enzyme
Catalase. Catalase enzyme is found in most bacteria. It catalyses
the breakdown of hydrogen peroxide (H2O2) with the release of
free Oxygen. Catalase is found in most aerobic and facultative
anaerobic bacteria.
Reagent -
3% H2O2 .
Procedure-
1. The sterile glass slide was taken.
2. 1 drop of 3% H2O2 was placed on slide and the single colony
was mixed with sterile loop.
3. The slide was observed for immediately and vigorous
bubbling.
4. A positive result was the rapid evolution of O2 as evidenced
by bubbling.
5. A negative result was no bubbles or only a few scattered
bubbles.
55
2. NITRATE TEST :
During anaerobic nitrate respiration Bacillus subtilis reduces
nitrate via nitrite to ammonia. No denitrification products were
observed. B. subtilis wild-type cells and a nitrate reductase
mutant grew anaerobically with nitrite as an electron acceptor.
NO3 ----> NO2 ----> NH3 or N2
Reagents -
Nitrate broth.
Sulfanilic.
Alpha-naphthylamine.
Powdered zinc.
PROCEDURE -
1.Inoculate separate tubes of nitrate broth with each of assigned
bacteria.
2. Incubate the tubes at 37°C for 24-48 hours.
3. After incubation, add five drops of sulfanilic acid and then five
drops of alpha- naphthylamine to each tube.
4. Observe whether or not a red coloration develops in the
cultures. The development of a red color indicates the reduction
of nitrates to nitrites. If no color develops, either the bacterium
56
cannot reduce nitrates to nitrites OR any nitrites produced were
rapidly further reduced to ammonia or other end products (that
would not impart the red color).
5. To determine if nitrites were produced, but then some or all
were reduced past the nitrite stage, add a minute quantity of
powdered zinc to any tubes that are colorless after the sulfanilic
acid and alpha-naphthylamine were added.
6. If a red color then appears after the addition of the zinc, this is
interpreted as NO reduction of nitrates (can't tell if the other
result, further reduction of all nitrites, has occurred). The zinc
actually reduces the nitrates to nitrites, which then produce the
red color in the presence of the sulfanilic acid and alpha-
naphthylamine.
57
3. OXIDASE TEST :
The oxidase test identifies organisms that produce the enzyme
cytochrome oxidase. Cytochrome oxidase participates in the
electron transport chain by transferring electrons from a donor
molecule to oxygen. The oxidase reagent contains a compound
that changes color when it becomes oxidized. If the test organism
produces cytochrome oxidase, the colorless reagent used in the
test will detect the presence of the enzyme oxidase and, reacting
with oxygen, turn violet to purple.
Reagent –
N, N, N`N`-Tetra methyl-p-phenylenediamine dihydrochloride.
(C6H4 [N (CH3)2]2.2HCl).
PROCEDURE –
1. Take 2-3 drops of (C6H4 [N (CH3)2]2.2HCl) oxidant on separate
slides. Using aseptic technique, Inoculate culture of assigned
bacteria on slides and mixed it.
3. Observe for the presence or absence of a color Change from
pink to maroon and finally to purple (lower portion of the plate).
If the change occurs in 10-30 seconds after adding the reagent,
the bacterium is considered positive for oxidase enzyme activity.
If no color change takes place, or the change is a slightly darker
pink, the bacterium is considered negative for oxidase activity.
58
4 .STARCH HYDROLYZING TEST:
Determines of a bacterium can hydrolyze starch (a
polysaccharide) into maltose and glucose. This is not a test to see
if the bacterium can ferment either sugar, only if it can hydrolyze
starch.
Reagents –
Starch agar.
Gram’s iodine.
1. Using aseptic technique, inoculate separate sections of plates
of starch agar with each of assigned bacteria.
2. Incubate the plates at 37oC for 24-48 hours.
3. Drip a small amount of Gram’s iodine on the plate around the
inoculated area, and a small amount in an uninoculated area
away from the inoculum.
4. If starch has been hydrolyzed, a clear zone will form around the
inoculum. If starch has not been hydrolyzed, no clear zone will
form and a blue-black color will result. The iodine reacts with
unhydrolyzed starch to produce the color.
5. Compare the inoculate area with the uninoculated area, and
record and interpret the results for assigned bacteria.
59
ISOLATION OF DNA
Reagents and Solutions:-
T.E Buffer (pH 8.0)
o 0.1M Tris HCl
o 0.01M EDTA
5M NaCl (29.3g of NaCl was dissolved in 1000ml of distilled
water, autoclaved and stored at room temperature).
CTAB/NaCl (4.1g NaCl and 10g CTAB was dissolved in 1000 ml
distilled water at 650C and stored at temperature).
Chloroform/Isoamyl alcohol (mix 24 volume of chloroform with
1 volume of isoamyl alcohol (24:1). It should be prepared
fresh).
10% SDS (10g SDS was dissolved in 100 ml distilled water by
heating at 650C in water bath for 20 min. do not autoclaved,
stored at room temperature).
Lysozyme (20mg lysozyme was dissolved in 1ml deionized
distilled water. The solution is stored in small aliquots at –
200C)
60
Proteinase-k (10mg of proteinase was dissolved in 1ml
deionized distilled water and the solution is stored at –200C).
70% Ethanol.
Isopropanol.
PROCEDURE:-
1 or 2 loops full of microbial growth was scraped from
culture media and suspended into 400 μl of T.E .buffer in a
vial.
The vial was freezed and thaw by –200C for 15 minutes
and heated it immediately up to 80 – 1000C for 5 min. and
again snap cooled at by keeping the vial in ice for 15 min.
The step was repeated.
40 µl lysozyme was added in the vial, mixed well and incubate
for 2 hours at 370C in shaking water bath.
56 µl of 10% SDS and 5 µl of proteinase –k was added in the
vial, mixed well and incubated at 65oC in shaking water bath
for 30 minutes.
61
80 µl of 5M NaCl and 64 μl of CTAB/NaCl solution were added
in the vial and incubate at 650C in water bath for 30 minutes.
Equal volume of freshly prepared Chloroform/Isoamyl alcohol
solution (24:1) was added in vial, mixed well and centrifuge at
10,000 rpm for 15 minutes. Three layers become visible. The
upper aqueous layer contains DNA, which is taken into
another fresh micro centrifuge tube.
0.6 volume of Isopropanol was added in vial in the
supernatant and incubated at –200C for 30 minutes.
The tube was centrifuged at 8000xg (10,000rpm) for 5
min.
The supernatant was discarded without losing pellet.
150 µl of 70% chilled ethanol was added in tube and
centrifuge the tube at 8000xg (10,000rpm) for 5 min.
The supernatant was discarded and air dried the pellet.
The white pellet observed after centrifuged the tube.
30 µl d/w. was added in the tube and stored at –200C till use.
62
AGAROSE GEL ELECTROPHORESIS
Chemicals and Reagents:-
Tris Acetate EDTA Buffer(TAE Buffer) 50X :-
Tris base: 242g
Glacial Acetic Acid: 37.1ml
EDTA: 37.2g
63
The final volume was made up to 1000 ml with deionised distilled
water. pH was maintained up to 8.0, autoclaved at 1210C and
stored at room temperature.
Ethidium bromide dye :-
Ethidium bromide 10 mg
Distilled water 1ml
Agarose Gel (2%):-
Agarose 0.8 g
50X TAE 0.8 ml
Ethidium bromide dye 3 µl
Distilled water 39.2 ml
DNA loading dye:-
Bromo Phenol Blue 0.25%
Xylene cynol 0.25%
Glycerol 30%
The dye was prepared in d.w. and it should be stored at 4oc.
PROCEDURE:-
64
2% Agarose was dissolved in TAE Buffer.
The solution was boiled in a water bath mixing occasionally by
swirling with hands.
Agarose gel was boiled gently till it dissolved.
The solution was cooled up to 55oC and Ethidium bromide
(0.5/ml) was added into the solution and the solution was
dispensed in casting tray with appropriate well forming comb and
was allowed to solidify.
250 ml TAE Buffer was poured in electrophoretic unit.
Prepared gel was placed in such a way that the wells are
towards cathode. The sample were loaded in wells and run the gel
at 32V for 2 hours.
The gel was observed on U.V. Transilluminator.
PCR (POLYMERASE CHAIN REACTION)
Reagent & chemicals
Distilled water :
-
276.5 µl
10x PCR buffer :
-
35.0 µl
dNTPs 200 μM :
-
7.0 µl
primer(forward ) : 7.0 µl
65
-
primer(Reversed) :
-
7.0µl
Taq DNA
polymerase
:
-
3.5 µl
DNA sample :
-
2.0 µl
Sequence of Primer-
EM1F: 5’-GACAAGAGAAATTTCTACGAGCAAGTACAAT-3’
EM1R: 5’-GCAGCCTTCCAATTACTCCTTCTGCCACAGT-3’
PCR –cycle -
Initial denaturation at 940C for 5min. following 45 cycles with
denaturation at 940C for 1 min, annealing at 550C for 1 min,
extension at 720C for 1 min the final extension at 720C for 10 min.
PROCEDURE-
1. The master mix was prepared by mixing all the components
given above. This was done on ice. then 48µl of master mix
were added in each 6 PCR tubes.
66
2. DNA template 2 µl was added in PCR tubes and the tubes were
placed in thermocycler and the program was set and started
with the appropriate temperatures, time and number of cycles.
3. The PCR product was stored at -200C till use.
67
PLANT COLLECTION
Six plants extract were assayed against E. coli, Staphylococcus
aureus and Bacillus subtilis. Medicinal plants used for herbal
extract sensitivity are listed below in table 1.
Table 1
S.n
o
Common
Name
Botanical
Name
Parts
Used
Aqueou
s
Ethanoli
c
Methanoli
c
1. Peepal Ficus
religiosa
Leave
s
A-1 E-1 M-1
2. Guava P. guajava Leave
s
A-2 E-2 M-2
3. Neem A.indica Leave
s
A-3 E-3 M-3
68
4. Tulsi Ocimum
sanctum
Leave
s
A-4 E-4 M-4
5. Banyan
Tree
Ficus
benghalen
sis
Leave
s
A-5 E-5 M-5
6. Ashoka Saraca
asoca
Leave
s
A-6 E-6 M-6
7. Pomegrana
te
P.
granatum
Leave
s
A-7 E-7 M-7
We observed the effect of these 7 plants species against
Bacillus cereus
PREPARATION OF PLANT EXTRACTS
The plant parts to be used were washed in tap water and allow
drying.
It was further wiped with 70 % alcohol again dry it.
Grind the plant parts with the help of pristle and mortar.
Take 0.5 gram of each powder form of plant and dissolve it into
5 ml of ethanol, 5 ml of methanol and 5 ml of distilled water.
69
These plant extracts were centrifuged at 10,000 rpm for 10
minutes.
Transfer the supernatants into other tubes and discard the
pellet.
Store it into refrigerator still use.
METHOD OF SCREENING :
DISC DIFFUSION METHOD
The disc of Whatmann filter paper no. 1 were cut in 5mm diameter
and the stock solution 0.5gm/5ml aqueous, ethanolic and
methanolic concentration was made by dissolving 0.5gm of each
plant extract in 5ml of methanol, 5 ml of ethanol and 5 ml of
distilled water and 10µl of stock solution was poured on to the
discs and sterilized in hot air oven for 3 hour till 3 days. Store discs
at room temperature till use.
70
Then the discs were placed on to the Muller Hinton agar
medium keeping proper distance among disc, to check the effect of
these plant extracts against E. coli, S. aureus and B. subtilis.
PREPARATION OF MEDIA
[MUELLAR HINTON AGAR ]
Composition gm/liter
Beef infusion :-300
Casein acid hydrolysate :-17.50
Starch :-1.50
Agar :-17.00
pH :-7.3-0.2
PROCEDURE
All the ingredients were taken in the flask, stirred well to
dissolve.
The pH was adjusted to 7.3-0.2 by adding NaCl or HCl.
This medium was dispensed into culture flasks, autoclaved
at 121oC at 15 lb pressure for 15 min.
Allowed to cool at room temperature and poured in 9 Petri
dish.
71
All 9 petri dishes should be marked for E. coli, S. aureus and
B. subtilis and for aqueous, ethanolic and methanolic
separately.
Allow media to solidify
After solidification the medium was spread with pure culture
E. coli, S. aureus and B. subtilis .
Dip a sterile cotton swab in the standardized bacterial
suspension and spread evenly on the surface of Mueller
Hinton agar medium to inoculate it.
Allow the medium to dry for 5min.
Place the test antibiotic disc with a positive control on the
surface of the medium with the help of sterile forceps or
mechanical dispenser.
Incubate the Petri dishes at 35oC-37oC for 24 hrs.
Carefully observe for antibiotic sensitivity of the
microorganism and measure the zone of complete growth
inhibition around each antibiotic disc with the help of a
caliper or transparent plastic ruler.
72
STATISTICAL CRITERIA FOR INDICATION OF
EFFICACY
A statistical presentation of crude extract was classified on the
basis of inhibition zone. The effectiveness of microorganism is
further divided into four categories, Traces, Weak, Normal and
excellent. 1-3mm was included under Traces, more than 3 to 6 mm
was included under Weak, more than 6 to 9 mm is included under
Normal, more than 9 to12 or more included in excellent category.
The criterion is as follows:
Table 2
S.NO. Number of plant extract on
the basis of inhibition zone
Code Indication of
efficacy
1. 1-3 mm inhibition zone A Traces
2. More than 3 to 6mm
inhibition zone
B Weak
3. More than 6 to 9mm
inhibition zone
C Normal
73
4. More than 9 to 12mm
inhibition zone
D Excellent
RESULTS
5 sample were collected from different regions and cultured on
nutrient agar media and then on Bacillus differential agar media
which were tested through various biochemical test for the
identification of Bacillus cereus.
S.
No.
Area of
Collection
Sampl
e
Code
Type
of
Sampl
e
Colony
Colour
Gram’
s
Stain
Endospo
re
Stain
1. Shastripura
m,
Agra
R1Soil
White
+
Yellow
+ve +ve
2. Runkata,
Agra
R2Soil
White
+
Yellow
+ve +ve
3.Keetham ,A
gra
R3Soil
White +ve +ve
74
4.Rambagh ,
Agra
R4Soil
White +ve +ve
5.Sikandra ,A
gra
R5Soil
White +ve +ve
Fig - Different colonies of B. subtilis & B.cereus
Grown on Bacillus differential media
Table-2; Data of Biochemical Tests –
75
S.No
.
Samp
le
Code
Catala
se
Test
Nitra
te
Test
Oxida
se
Test
Starch
Hydrolyzing
Test
1. R-1 +ve +ve +ve +ve
2. R-2 +ve +ve +ve +ve
3. R-3 +ve +ve +ve +ve
4. R-4 +ve +ve +ve +ve
5. R-5 +ve +ve +ve +ve
Out of the 5 collected samples all were identified as B.cereus,
through biochemical tests.
BIOCHEMICAL TEST RESULTS -
1. CATALASE TEST
76
2. OXIDASE TEST
3. NITRATE REDUCTION TEST
77
4. STARCH HYDROLYSING TEST
OBSERVATIONS OF PCR FOR EMETIC TOXIN PRODUCING B.
CEREUS
Gel Electrophoresis of PCR Amplification
78
Hemolysis on Blood Agar
Lane-1: Marker (M)
Lane-2: Sample no.1 (R1)
Lane-3: Sample no.2 (R2)
Lane-4: Sample no.3 (R3)
Lane-5: Sample no.4 (R4)
Lane-6: Sample no.5 (R5)
Table-3; Data Of PCR emetic toxin producing B.
cereus
Results
S.No. Sample Code PCR result
1. R1 Amplified
2. R2 Amplified
3. R3 Amplified
4. R4 Not Amplified
5. R5 Amplified
79
Out of 5 samples only 4 samples (R1, R2, R3, R5,) identified as B.
cereus amplified through PCR which confirms the presence of B.
cereus at molecular level.
OBSERVATIONS OF PLANT EXTRACTS
AGAINST B. CEREUS
80
Fig: Antibiotic Discs on B. subtilis culture
Table-4; Data Of Zone of growth inhibition in (mm)
After 24 hours size of zone of growth inhibition measured in mm is given the following table:-
Plant
Code
Name Parts used
Zone of growth inhibition (in mm)
B. cereus
1. Peepal Bark 28
2. Guava Leaf 23
3. Neem Leaf 23
4. Tulsi Leaf 25
5. Banyan tree Leaf 28
6. Ashoka Leaf 30
81
7. Pomegranate Leaf 25
8. Norfloxacin Control 26
Out of the 7 plant extracts tested for antibacterial activity, all
plants extracts showed antibacterial activity by inhibiting Bacillus
cereus. Norfloxacin was taken as a positive control.
In this study all plants extracts are prepared in distilled water.
Among the plants screened, all plants showed promising activity
against Bacillus cereus.
DISCUSSION & CONCLUSION
Tables 1, 2, and 3 show that, using cultural characteristics, and
biochemical characteristics, of B. cereus. It is ubiquitous,
saprophytic, soil bacterium and its ability to produce a wide
variety of enzymes. This latter feature of the microorganism has
been commercially exploited for over a decade. B. cereus has
been used for industrial production of proteases, amylases,
antibiotics, and specialty chemicals.
One of the degradative enzymes synthesized early in
stationary phase in B. cereus alpha-amylase, an exo-enzyme
responsible for the degradation of starch to simpler sugars which
can be assimilated by the cell (Yamaguchi et al., 1974).
82
We have identify a gene of an extra cellular -amylase from
the mesophilic strain of B. cereus. The extra cellular -amylase
enzyme is not very closely related to any other amylases of family
13 of glycosyl hydrolases. On the other hand it can be aligned to
the other enzymes, and it has the conserved regions I-IV found in
other amylases.
The use of B. cereus in an industrial setting should not pose
an unreasonable risk to human health or the environment. First,
human health and environmental hazards of B. cereus are low.
Second, the number of microorganisms released from the
fermentation facility is low. In addition, B. cereus is ubiquitous in
the environment, and the releases expected from the
fermentation facilities will not significantly increase populations of
this bacterium in the environment.
The B. cereus genome contains several genes that are
predicted to code for proteins that belong to the cupin super
family. Cupins are proteins that are related to plant seed storage
proteins that fold into small beta-barrels. Several of the B. cereus
cupins share identity with the secreted oxalate-degrading
enzymes of fungi and plants. Its genome of 4,214,810 base pairs
comprises 4,100 protein-coding genes.
In addition, the availability of the complete genome
sequence (Kunst, F, 1997) and about 3,000 "y"-mutants
constructed within the B. cereus Functional Analysis program
83
(Kobayashi K 2003, Schumann, W., 2000) make B. cereus an
ideal model organism for research on gram-positive bacteria.
Plants are important source of potentially useful structures
for the development of new chemotherapeutic agents. The first
step towards this goal is the in vitro antibacterial activity assay
(Tona, L., K. Kambu, N. Ngimbi, K. Cimanga and A.J.
Vlietinck, 1998, ). Many reports are available on the antiviral,
antibacterial, antifungal, anthelmintic, antimolluscal and anti-
inflammatory properties of plants (Samy, R.P. and S.
Ignacimuthu, 2000, Palombo, E.A. and S.J. Semple, 2001).
Some of these observations have helped in identifying the active
principle responsible for such activities and in the developing
drugs for the therapeutic use in human beings. However, not
many reports are available on the exploitation of antibacterial
property of plants for developing commercial formulations for
applications in crop protection. In the present study, the methanol
leaf, root/bark extracts of Acacia nilotica, Tinospora cordifolia,
Withania somnifera and Ziziphus mauritian showed the activity
against B. cereus.
The results of present investigation clearly indicate that the
antibacterial and antifungal activity vary with the species of the
plants and plant material used. Thus, the study ascertains the
value of plants used in ayurveda, which could be of considerable
interest to the development of new drugs.
84
In conclusion, the use of B. cereus in fermentation facilities
for the production of enzymes or specially chemicals has low risk.
Although not completely innocuous, the industrial use of B. cereus
presents low risk of adverse effects to human health or the
environment.
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