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:

1

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.

2

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

3

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

4

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.

5

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

6

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,

7

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

8

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

9

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

10

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.

11

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

12

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

13

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

15

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.

16

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.

18

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].

19

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

20

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

21

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

22

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].

23

Fig4: Different type of B.cereus

Fig5: Electron microscopic Structure of

Flagella

24

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,

25

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

26

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

27

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.

28

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

29

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).

30

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

31

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.

REFERENCES

1. Aas, N. Gondrosen, B. and Langeland, G. (1992). Norwegian

Food Authorities Report on Food Associated Diseases in 1990.

SNT Report Oslo.

2. Abram D., Vatter, A. E. and Koffler, H. (1966). Attachment and

structural features of flagella of certain bacilli. J. Bacteriol. 95:

2045-2068.

3. Agata N, Mori M, Ohta M, Suwan S, Ohtani I, Isobe M (1994). A

novel dodecadepsipeptide, cereulide, isolated from Bacillus

85

cereus causes vacuole formation in HEp-2 cells. FEMS Microbiol

Lett., 121: 31–34.

4. Agata N, Ohta M, Mori M, Isobe M (1995). "A novel

dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus

cereus". FEMS Microbiol Lett, 129 (1): 17–20

5. Agata, N., Ohta, M., Mori, M. and Isobe, M. (1995) A novel

dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus

cereus. FEMS Microbiol. Lett. 129: 17-20.

6. Allison, C., P. Jones, N. Coleman, and C. Huges. (1992). Ability

of Proteus mirabilis to invade human urothelial cells is coupled

to motility and swarming differentiation. Infect. Immun. 60:

4740-4746.

7. Andersson, M., Mikkola, R., Helin, J., Andersson, M.C. and

Salkinoja Salonen, M. (1998). A novel sensitive bioassay for

detection of B. cereus emetic toxin and related depsipeptide

ionophores. Appl. Environ. Microbiol. 64: 1338-1343.

8. Aviram, M., Dornfeld, L. (2001). "Pomegranate juice

consumption inhibits serum angiotensin converting enzyme

activity and reduces systolic blood pressure". Atherosclerosis.

158 (1): 195–8

9. Aviram M, Rosenblat M, Gaitini D. (2004). "Pomegranate juice

consumption for 3 years by patients with carotid artery

stenosis reduces common carotid intima-media thickness,

blood pressure and LDL oxidation". Clin Nutr., 23 (3): 423–33.

86

10. Beveridge, T. J. (1997). The response of S-layered bacteria to

the Gram stain. FEMS Microbiol Rev.20: 101–110.

11. Drobniewski, F.A. (1993). Bacillus cereus and related

species. Clin. Microbiol. Rev. 6: 324-338.

12. Ellar, D. J. and Lundgren, D.G. (1967). Ordered structure in

the cell wall of Bacillus cereus. J Bacteriol. 94: 1778–1780

13. Finlay, W.J., Logan, N.A. and Sutherland, A.D. (1999).

Semiautomated metabolic staining assay for Bacillus cereus

emetic toxin. Appl. Environ. Microbiol. 65: 1811-1812.

14. Girisch, M., Ries, M., Zenker, M., Carbon, R., Rauch, R. and

Hofbeck, M. (2003). Intestinal perforations in a premature

infant caused by Bacillus cereus. Infection 31: 192-193.

15. Granum, P.E. (1994). Bacillus cereus and its toxins. J. Appl.

Bacteriol. 23 (Symp. Suppl.), 61S-66S.

16. Granum, P.E. (2001). Bacillus cereus. In: Food Microbiology:

Fundamentals and Frontiers (Doyle, M.P. et al., Eds.), pp. 373-

381. ASM Press, Washington, D.C.

17. Guinebretiere, M.H., Broussolle, V. and Nguyen-The, C.

(2002). Enterotoxigenic profiles of food-poisoning and food-

borne Bacillus cereus strains. J. Clin. Microbiol. 40: 3053-3056.

18. Becker, H., Schaller, G., Von Wiese, W., Terpian, G. (1994).

Int. J. Food Microbiol. 23: 1–15.

19. Haggblom, M.M., Apetroaie, C., Andersson, M.A. and

Salkinoja- Salonen, M.S. (2002). Quantitative analysis of

87

cereulide, the emetic toxin of Bacillus cereus, produced under

various conditions. Appl. Environ. Microbiol. 68: 2479-2483.

20. Hansen, B.M. and Hendriksen, N.B. (2001). Detection of

enterotoxic Bacillus cereus and Bacillus thuringiensis strains by

PCR analysis. Appl. Environ. Microbiol. 67: 185-189.

21. Hocking, A.D. et al. (1997). Foodborne Microorganisms of

Public Health Significance. 5th ed. North Sydney. AIFST NSW

Branch Food Microbiology Group.

22. Kramer, J. M., Gilbert, R. J. (1989). Bacillus cereus and other

bacilli species. In: Food-Borne Pathogens, M. P. Doyle (Ed.),

Marcel Dekker, New York pp. 21–70.

23. Jimenez, E.A., Rincon, M., Pulido, R., Saura, C.F. (2001).

Guava fruit (Psidium guajava) as a new source of antioxidant

dietary fiber. Journal of Agricultural and Food Chemistry.49(11):

5489–5493

24. Jo, C., Lee, N.Y., Kang, H.J., Shin, D.H. and Byun, M.W.

(2004). Inactivation of food-borne pathogens in marinated beef

rib by ionizing radiation. Food Microbiol. 21: 543–8

25. Kevin, M. Devine, et al. (2008). A matter of life and death:

cell wall homeostasis and the WalKR (YycFG) essential signal

transduction pathway. Mol. Microbiol.

26. Kotiranta, A., Lounatmaa, K., Haapasalo, M. (2000).

"Epidemiology and pathogenesis of Bacillus cereus infections".

Microbes Infect 2 (2): 189–98.

88

27. Lawrie, R.A., (1998). Lawrie’s Meat Science (6th ed.). Wood-

head Publishing Limited, Cambridge.

28. Mahfuzul Hoque, M.D., Bari, M.L., Inatsu, Y., Juneja, V.K., and

Kawamoto, S. (2007). Antibacterial activity of guava (Psidium

guajava) and Neem (Azadirachta indica A. Juss.) extracts

against food borne pathogens and spoilage bacteria.

Foodborne Pathogens and Disease. 4(4): 481–488

29. Mahler, H., Pasi, A., Kramer, J.M., Schulte, P., Scoging, A.C.,

Bar, W. and Krahenbuhl, S. (1997). Fulminant liver failure in

association with the emetic toxin of Bacillus cereus. New Engl.

J. Med. 336: 1142-1148.

30. McCarter, L., Hilmen, M. and Silverman, M. (1988). Flagellar

dynamometer controls swarmer cell differentiation of V.

parahaemolyticus. Cell. 54: 345-351

31. McKillip, J.L. (2000). "Prevalence and expression of

enterotoxins in Bacillus cereus and other Bacillus spp., a

literature review". Antonie Van Leeuwenhoek. 77 (4): 393–9.

32. Melendez P.A., Capriles V.A. (2006). Antibacterial properties

of tropical plants from Puerto Rico. Phytomed. 13: 272-276.

33. Menezes, S.M., Cordeiro, L.N., Viana, G.S. (2006). "Punica

granatum (pomegranate) extract is active against dental

plaque". Journal of herbal pharmacotherapy. 6 (2): 79–92.

34. Messner, P., Sleytr, U.B. (1992). Crystalline bacterial cell-

surface layers. Adv Microbiol Physiol. 33: 214–275.

89

35. Mikkola, R., Saris, N.E., Grigoriev, P.A., Andersson, M.A. and

Salkinoja Salonen, M.S. (1999). Ionophoretic properties and

mitochondrial effects of cereulide: the emetic toxin of B.

cereus. Eur. J. Biochem. 263: 112-117.

36. Mosupye, F.H. and A. Von Holg, (2000). Microbiological

hazard identification and exposure assessment of street food

vending in Johannesburg, South Africa. Int. J. Food Microbiol.

61: 137–45

37. National Committee for Clinical Laboratory Standards (1999).

Molecular Diagnostic Methods for Infectious Diseases.

Approved Guideline MM3-A. NCCLS, Wayne, PA.

38. Noanao, B. and Trust, T. J(1997). The synthesis, secretion

and role in virulence of the paracrystalline surface protein

layers of Aeromonas salmonicida and A. hydrophila. FEMS

Microbiol Lett. 154: 1–7.

39. Nortje, G.L., Vorster, S.M., Greebe, R.P. and Steyn, P.L.

(1999). Occurrence of Bacillus cereus and Yersinia

enterocolitica in South African retail meats. Food Microbiol., 16:

213–7

40. Notermans S, Dufrenne, J., Teunis, P., Beumer, R., Te Giffel,

M., Peeters Weem, P. (1997): Food Microbiol. 14: 143–151.

41. Paananen, A., Mikkola, R., Sareneva, T., Matikainen, S., Hess,

M., Andersson, M., Julkunen, I., Salkinoja-Salonen, M.S.,

Timonen, T. (2002). Inhibition of human natural killer cell

activity by cereulide, an emetic toxin from Bacillus cereus. Clin

Exp Immunol. 129: 420–428.

90

42. Paananen, A., Mikkola, R., Sareneva, T., Matikainen, S., Hess,

M., Andersson, M., Julkunen, I., Salkinoja-Salonen, M.S. and

Timonen, T. (2002). Inhibition of human natural killer cell

activity by cereulide, an emetic toxin from Bacillus cereus. Clin

43. . Exp. Immunol. 129: 420-428.

44. Pru, B.M., Dietrich, R., Nibler, B., Martlbauer, E. and Scherer,

S. (1999). The hemolytic enterotoxin HBL is broadly distributed

among species of the Bacillus cereus group. Appl. Environ.

Microbiol. 65: 5436-5442.

45. Satin, M., (2002). Use of irradiation for microbial

decontamination of meat: situation and perspectives. Meat Sci.

62: 277–83

46. Schaechter, M., Ingraham, J.L., Neidhardt, F.C. (2006).

Microbe. (ASM Press, Washington, DC,).

47. Schmidt, K. and Tirado, C. (2001) WHO Surveillance Program

for Control of Foodborne Infections and Intoxications in Europe

(Seventh report 1993-1998), p. 110. WHO, Berlin.

48. Seeram, N.P., Aronson, W.J., Zhang, Y., et al. (2007).

"Pomegranate ellagitannin-derived metabolites inhibit prostate

cancer growth and localize to the mouse prostate gland". J.

Agric. Food Chem. 55 (19): 7732–7735.

49. Shinagawa, K., Konuma, H., Sekita, H. and Sugii, S. (1995)

Emesis of rhesus monkeys induced by intragastric

administration with the HEp-2 vacuolation factor (cereulide)

produced by Bacillus cereus. FEMS Microbiol. Lett. 130: 87-90.

91

50. Srivastava, J., Lambert, J. and Vietmeyer, N. (1996).

Medicinal plants: An expanding role in development. World

Bank Technical Paper. No. 320.

51. Stachelhaus, T. and Marahiel, M.A. (1995). Modular structure

of genes encoding multifunctional peptide synthetases required

for non-ribosomal peptide synthesis. FEMS Microbiol. Lett. 125:

3-14.

52. Stockwell, C. (1988). Nature’s pharmacy. London, United

Kingdom. Century Hutchinson Ltd.

53. Thomson, W.A.R., (1978). Medicines from the Earth.

Maidenhead, United Kingdom. McGraw-Hill Book Co.

54. Toba, T., Virkola, R., Westerlund, B., Bjorkman, Y., Sillanpa,

J., Vartio, T., Kalkkinen, N., Korhonen, T. K. (1995). A collagen-

binding S-layer protein in Lactobacillus crispatus. Appl. Environ

Microbiol. 61: 2467–2471.

55. Uniyal, S.K., Singh, K.N., Jamwal P. and Lal, B. (2006).

Traditional use of medicinal plants among the tribal

communities of Chhota Bhangal, Western Himalayan. J.

Ethnobiol. Ethnomed., 2: 1-14.

92

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