DECLARATION

I hereby declare that the work which has been presented in the dissertation entitled To check the activity of -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:

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 friends Ved, Anu, Archarna, Amita, for their affection & encouragement during the course of my study.

(Shweta Dass) B.E. biotechnology

3

TABLE OF CONTENTS

S. NO. 1 2 3 4 5 6 7 8

TITLE ABBRIVATION AIM OF STUDY INTRODUCTION REVIEW OF LITERATURE METHOD & MATERIALS RESULTS DISCUSSION & CONCLUSION REFFERENCE

PAGE NO. 5 6 7-11 12-44 45-69 66-69 70-77 78-88

ABBRIVATION
B . cereus gm Mg l ml rpm d/w UV LIGHT C EDTA TAE BUFFER TE BUFFER DNA Tm Bacillus Cereus gram milli gram micro litre milli litre revolution per minute distilled water ultra violet light degree centrigrate Ethylene diamine tetra acetic acid tris acetic acid EDTA buffer tris EDTA buffer Dioxy ribonucleic acid melting temperature

AIM OF STUDY
5

On considering the role of B-cereus in several diseases is selected the study To check the activity of -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. 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 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
7

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, Bacillus (written with a capital letter and italicized) refers to a specific genus of bacteria. The family Bacillaceae are all Grampositive, 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 m wide, 5-10 m 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 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 the small, heat- and acid-stable cyclic dodecadepsipeptide cereulide
[2] [3]

. While enterotoxins are comparatively well the molecular

[4]

characterized

at

and

the

expression

level

(Granum, P.E. 2001)

, 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 acting as a potassium ionophore
[5]

is toxic to mitochondria by and it was involved in

[7]

[6]

fulminant liver failure in a human case

. Recently, it has been

[8]

reported that cereulide inhibits human natural killer cells and might therefore have an immunomodulating effect .

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
10

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 causative agent was identified
[10] [9]

and it was

responsible for 14% of the outbreaks in Finland in which the . 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 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

11

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. 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 nongastrointestinal 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 quick12

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

[19]

complications of B. cereus infections in premature neonates might be at least partly related to enterotoxins . 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.

13

SCIENTIFIC CLASSIFICATION
Bergeys 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
14

include the Order Bacillales and the Family Bacillaceae. In this family there are 37 new genera on the level with Bacillus.

TAXONOMIC CLASSIFICATION

Kingdom Phylum Class Order Family Genus Species

:::::::-

Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus cereus

15

HISTORY

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 19471949, 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
16

the dessert had clinical symptoms of food poisoning. To provide evidence that B. cereus was the cause of food poisoning, Hauge demonstrated Kochs postulates by consuming a culture of the isolated B. cereus strain. He grew B. cereus to a level of 4106 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. In 1954, experiments with volunteers in USA failed to confirm Hauges 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 15 hrs after consumption of the incriminated meal. Sometimes, the incubation time was as short as 1530 min or as long as 612 hrs. Almost all the vomiting type outbreaks were associated with consumption of cooked rice. This type of poisoning resembled staphylococcal food poisoning.

17

SOURCES OF BACILLUS CEREUS

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

18

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.

4.Dairy products
Rice and cooked oriental foods Its 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
19

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

[22]

sources of Bacillus cereus contamination (Lawrie, 1998)

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]

20

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 gramnegative 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
21

collagen in Lactobacillus crispatus

[27]

Increased virulence and

[28]

resistance to phagocytosis (Noanao B, Trust T J 1997)

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
22

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
23

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 ;) are found to be composed of three
[34]

they

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]

24

Fig4: Different type of B.cereus

Fig5: Electron microscopic Structure of Flagella

25

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,
26

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 816.5 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 perfringens. from poisoning caused by Clostridium

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 15 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
27

types, but in fact the emetic syndrome is caused by a toxin called 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) reported to inhibit human natural killer cells
[39]

. It is toxic to . According to its

28

mitochondria by acting as a potassium ionophore and has been

[40]

chemical structure it has been shown that this toxin is produced 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 threecomponent 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.

29

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

30

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, (e.g. zwittermicin), Bacillus circulans polymyxa (e.g. (e.g. circulin), Brevibacillus laterosporus (e.g. laterosporin), Bacillus licheniformis bacitracin), Paenibacillus polymyxin, colistin), Bacillus pumilus (e.g. pumulin) and Bacillus subtilis (e.g. polymyxin, difficidin, subtilin, mycobacillin).

31

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 Division Class Oder Family Genus Species Scientific Name ::::::::Plantae Magnoliophyta Magnoliopsida Rosales Moraceae Ficus F.religiosa Ficus religiosa

32

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.

33

2.GUAVA Scientific classification:

Kingdom Division Class Subclass Oder Family Subfamily Genus Species Scientific Name Plantae Angiosperms Eudicots Rosids Myrtales Myrtaceae Myrtoideae Psidium P.guajava Psidium guajava

:::::::::-

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.

34

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,
35

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:

Kingdom Division Class Order Family Genus Species Scientific name :::::::Plantae Magnoliophyta Magnoliopsida Lamiales Lamiaceae Ocimum O. tenuiflorum Ocimum Sanctum

1. Fever & Common Cold:-The many malaria fever leaves of basil are specific for fevers. and are During dengue widely tender the rainy season, when

prevalent,

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
36

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.

37

4.NEEM TREE Scientific classification:

38

Kingdom Division Class Order Family Genus Species Scientific name

Planate :- Magnoliophyta :- Magnoliopsida :- Sapindales :- Meliaceae :- Azadirachta :- A. Indica :- Azadirachta indica 1. Fruit:-The fruit is a smooth olive-like which shape elongate nearly oval (glabrous) drupe varies in from to 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
39

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 1520 m (about 5065 feet), rarely to 3540 m (115131 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 2040 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 56 mm long and 811 mm wide. Protandrous, bisexual flowers and male flowers exist on the same individual.

5. BANYAN TREE Scientific classification:

40

Kingdom Division Class Order Family Genus Scientific Name

Plantae :- Magnoliophyta :- Magnoliopsida :- Urticales :- Moraceae :- Ficus :- 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.

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
41

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

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.

42

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 (kma) overcomes humans.[50]

43

6. SARACA ASOCA Scientific classification:

Kingdom Division Class Order Family Genus Species Scientific name :::::::Plantae Angiosperms Eudicots Fabales Fabaceae Saraca S. asoca Saraca asoca 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]

4. Then the tree magically bent down for her and she grasped a branch. At side.[52] that moment the Buddha emerged from her right

44

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:
Kingdom Division Class Subclass Order Family Genus Speces Plantae Magnoliophyta Magnoliopsida Rosidae Myrtales

::::-

:- Lythraceae :- Punica :- P. Granatum Punica granatum

Scentific Name :-

1.

pomegranate

(Punica

granatum)

is

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 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, 37 cm long and 2 cm broad. The flowers
45

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, 512 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]

46

5. Health Benefits:- In preliminary laboratory research and clinical trials, juice of the pomegranate may be effective in reducing formation. heart
[56][57][58]

disease

risk

factors,

including

LDL

oxidation, macrophage oxidative status, and foam cell 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
[60]

enzyme.[59] Juice consumption may also inhibit viral infections dental plaque.[61]
[62]

while pomegranate extracts have antibacterial effects against

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

47

WASHING 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 C 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 C 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. 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.

To dry the Tip we use the Hot air oven at 37 C for 30 min.

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.
48

Set the Autoclave at 121 C for 15 min. And Pressure was 15 Psi.

Store the wrapped glassware for further use. 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 C for 15 min. And Pressure was 15 Psi. Sterilize the platinum loop by the Flame (direct heat).Whenever the loop was red hot.

Store the wrapped boxes for further use.

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.

49

SAMPLE COLLECTION:5 samples were collected from different region of Agra.

S.No .
1. 2. 3. 4. 5.

Area of Collection
Shastripuram , Agra Runkata , Agra Keetham ,Agra Rambagh , Agra Sikandra ,Agra

Type of Sample
Soil Soil Soil Soil Soil

Type of Sample
R1 R2 R3 R4 R5

50

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 80C 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 Peptic digest tissue Beef extract Yeast extract Sodium chloride Agar Final pH (at 25C) gm/liter 5.00 1.50 1.50 5.00 15.00 7.4 0.2

of

animal ::::::-

PROCEDURE-

51

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

Bromo cresol purple Agar Final pH (at 25C) :::0.0075 15.40 72

All the ingredients were taken in the flask, stirred well to dissolve. The pH was adjusted to 7.40.2 by adding NaCl or HCl.
52

 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.GRAMS STAINING: ReagentsGrams stain Moderant Decolorizing agent Counter stain :- Crystal Violet :- Grams Iodine :- 70% Alcohol :- Safranin

Procedure:

The smear was prepared on sterilized glass slide. The smear was fixed by passing over the flame.

53

The

smear

was

flooded

with

crystal

violet

and

incubated for 2 min.

The smear was washed with tap water. The smear was flooded with grams 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

Grams stain Counter stain Crystal Violet 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.

54

Remove blotting paper and rinse slide with water until Flood slide with the counterstain safranin for 20 View specimen under oil immersion lens with light

water runs clear.

seconds and then rinse.

microscope.

BIOCHEMICAL TEST
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 .

Procedure55

1.
2.

The sterile glass slide was taken. 1 drop of 3% H2O2 was placed on slide and the single The slide was observed for immediately and vigorous A positive result was the rapid evolution of O2 as A negative result was no bubbles or only a few

colony was mixed with sterile loop. 3. bubbling.

4.

evidenced by bubbling. 5. scattered bubbles.

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.

56

Powdered zinc.

PROCEDURE 1.Inoculate separate tubes of nitrate broth with each of assigned bacteria. 2. Incubate the tubes at 37C 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 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

57

red color in the presence of the sulfanilic acid and alphanaphthylamine.

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
58

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.

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. Grams iodine. 1. Using aseptic technique, inoculate separate sections of plates of starch agar with each of assigned bacteria.
59

2. Incubate the plates at 37oC for 24-48 hours. 3. Drip a small amount of Grams 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.

ISOLATION OF DNA
Reagents and Solutions: o o

T.E Buffer (pH 8.0) 0.1M Tris HCl 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).
60

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) Proteinase-k (10mg of proteinase was dissolved in 1ml deionized distilled water and the solution is stored at 200C). 70% Ethanol. Isopropanol.

PROCEDURE: vial.

1 or 2 loops full of microbial growth was scraped from culture media and suspended into 400 l of T.E .buffer in a 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 l lysozyme was added in the vial, mixed well and incubate for 2 hours at 370C in shaking water bath.

61

56 l of 10% SDS and 5 l of proteinase k was added in the vial, mixed well and incubated at 65oC in shaking water bath for 30 minutes.

80 l of 5M NaCl and 64 l 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. min.

The tube was centrifuged at 8000xg (10,000rpm) for 5 The supernatant was discarded without losing pellet. 150 l 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 l 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: Glacial Acetic Acid: EDTA: 242g 37.1ml 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 Distilled water 10 mg 1ml

Agarose Gel (2%):Agarose 50X TAE Ethidium bromide dye Distilled water 0.8 g 0.8 ml 3 l 39.2 ml

DNA loading dye:Bromo Phenol Blue Xylene cynol Glycerol 0.25% 0.25% 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 10x PCR buffer dNTPs 200 M primer(forward ) primer(Reversed) : : : : : 65

276.5 l 35.0 l 7.0 l 7.0 l 7.0l

Taq polymerase DNA sample

DNA : : -

3.5 l 2.0 l

Sequence of PrimerEM1F: 5-GACAAGAGAAATTTCTACGAGCAAGTACAAT-3 EM1R: 5-GCAGCCTTCCAATTACTCCTTCTGCCACAGT-3

PCR cycleInitial 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.

PROCEDURE1. The master mix was prepared by mixing all the components given above. This was done on ice. then 48l of master mix were added in each 6 PCR tubes. 2. DNA template 2 l 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.

66

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 1. Common Name Peepal Botanical Name Ficus religiosa Parts Aqueou Ethanoli c E-1 Methanolic M-1

Used s Leave A-1 s

67

2. 3. 4. 5.

Guava Neem Tulsi Banyan Tree

P. guajava Leave A-2 A.indica Ocimum sanctum Ficus benghale nsis Saraca asoca P. granatum s Leave A-3 s Leave A-4 s Leave A-5 s Leave A-6 s Leave A-7 s

E-2 E-3 E-4 E-5

M-2 M-3 M-4 M-5

6. 7.

Ashoka Pomegrana te

E-6 E-7

M-6 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.

68

 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 10l 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. 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
69

Beef infusion :Casein acid hydrolysate :Starch Agar pH :::-

300 17.50 1.50 17.00 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.

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.
70

Incubate the Petri dishes at 35oC-37oC for 24 hrs. 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.

Carefully

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
71

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 Code Indication the basis of inhibition zone 1. 2. 3. 4. 1-3 mm inhibition zone More More More than than than 3 6 9 to to to 6mm 9mm 12mm inhibition zone C D Normal Excellent inhibition zone inhibition zone A B efficacy Traces Weak of

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 Colony Colour Gram s Stain Endospor e Stain
72

1.

Shastripura m, Agra

R1

e Soil

White + Yellow White + Yellow White White White

+ve

+ve

2.

Runkata, Agra

R2

Soil

+ve

+ve

3. 4. 5.

Keetham ,Agra Rambagh , Agra Sikandra ,Agra

R3 R4 R5

Soil Soil Soil

+ve +ve +ve

+ve +ve +ve

73

Fig - Different colonies of B. subtilis & B.cereus Grown on Bacillus differential media

Table-2; Data of Biochemical Tests

S.No Samp . 1. 2. 3. 4. 5. le Code R-1 R-2 R-3 R-4 R-5

Catala se Test +ve +ve +ve +ve +ve

Nitra te Test +ve +ve +ve +ve +ve

Oxida se Test +ve +ve +ve +ve +ve

Starch Hydrolyzing Test +ve +ve +ve +ve +ve

Out of the 5 collected samples all were identified as B.cereus, through biochemical tests.

BIOCHEMICAL TEST RESULTS 74

1. CATALASE TEST

2. OXIDASE TEST

75

3. NITRATE REDUCTION TEST

4. STARCH HYDROLYSING TEST

76

Hemolysis on Blood Agar

OBSERVATIONS OF PCR FOR EMETIC TOXIN PRODUCING B. CEREUS

Gel Electrophoresis of PCR Amplification 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)

77

Table-3; Data Of PCR emetic toxin producing B. cereus Results

S.No. 1. 2. 3. 4. 5. Sample Code R1 R2 R3 R4 R5 PCR result Amplified Amplified Amplified Not Amplified Amplified

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

78

AGAINST B. CEREUS

Fig: Antibiotic Discs on B. subtilis culture

Table-4; Data Of Zone of growth inhibition in (mm)

79

After 24 hours size of zone of growth inhibition measured in mm is given the following table:Pla nt Cod e 1. 2. 3. 4. 5. 6. 7. 8. Name Parts used Zone of growth inhibition (in mm) B. cereus 28 23 23 25 28 30 25 26

Peepal Guava Neem Tulsi Banyan tree Ashoka Pomegranate Norfloxacin

Bark Leaf Leaf Leaf Leaf Leaf Leaf Control

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

80

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). 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.
81

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 (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 antiinflammatory 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
82

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. 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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