DECLARATION

I hereby declare that the work which has been presented in the dissertation entitled Isolation & characterization of B. cereus isolated from soil samples, identification of emetic toxin producing gene in B. cereus at molecular level and to check the antimicrobial activity of medicinal plants against B .cereus. 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, 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. for their valuable

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

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 7-10 11-40 40-63 63-71 71-74 74-82

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
On considering the role of B-cereus in several diseases we selected the study Isolation & characterization of B. cereus isolated from soil samples , identification of the emetic toxin producing gene in B. cereus at molecular level and to check the antimicrobial activity of medicinal plants against B .cereus with following objectives.

Isolation of B.cereus from different soil sample. Characterization of isolated B.cereus at Biochemical level. Identification of Emetic toxin producing strains of 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 milk, milk products , 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
7

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, 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, rodshaped bacteria which are ubiquitous in nature. Gram-stained cells, 1 m wide, 510 m long, arranged singly or in short chains. The organism produces heat resistant spores and these may germinate if cooling is too slow [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
8

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 characterized at the molecular and the expression level [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
9

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 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 nontoxic 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
10

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. 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
11

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

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TAXONOMIC CLASSIFICATION

Kingdom Phylum Class Order Family Genus Species

:::::::-

Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus cereus

13

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 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.
14

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.

15

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

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.

16

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
Microorganisms control in meat products is the major concern in the preparation of high quality foods [20]. The hygienic state of animals prior, during and after slaughter can be critical to the finished product quality [21]. During slaughtering process the meat is exposed to many sources of Bacillus cereus contamination [22]. The incidence of Bacillus cereus is higher in cooked and processed (ground beef) meat than in raw meat samples [23] [24].

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STRUCTURE OF BACILLUS CEREUS

Like most Gram-positive bacteria the surface of the Bacillus cereus is complex and is associated with their properties of adherence, resistance and tactical responses. The vegetative cell surface is a laminated structure that consists of a capsule, a proteinaceous surface layer (S-layer), several layers of peptidoglycan sheeting, and the proteins on the outer surface of the plasma membrane.

Fig2: Surface of a Bacillus cereus Transmission E.M. C=Capsule; S=S-layer; P=Peptidoglycan.

Surface layer (S-layer) :A regularly ordered protein or glycoprotein layer (S-layer) has been detected as the outermost component of several gram-negative and gram-positive organisms [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 collagen in Lactobacillus crispatus [27] Increased virulence and resistance to phagocytosis [28] have been associated with the presence of the S-layer in animal pathogens. Ellar and Lundgren [29] described the presence of an S-layer on the surface of B. cereus .
18

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. [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 mesodiaminopimelic 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 crosslinked to D-alanine, same as in the Enterobacteriaceae; in other cases, two tetrapeptide side chains of peptidoglycan are spanned by an interpeptide bridge 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.

19

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 [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 cytoplasmic compartments to coordinate the production of precursors/enzymes with their utilization. [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 [33] . When bacterial flagella are examined by electron microscopy [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.
20

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 [35].

Fig5: Electron microscopic Structure of Flagella

21

Fig4: Different type of B.cereus

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

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Pathogenesis of Bacillus cereus

B. cereus is responsible for a minority of food borne illnesses (25%), causing severe nausea, vomiting and diarrhea [36]. Generally speaking, 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 [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 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 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 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
23

peptide synthesis (NRPS), which is somewhat unusual in itself. Cereulide is believed to activate 5-HT receptors leading to increased afferent vagal stimulation [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 [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 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.
24

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.

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

25

between species, but with the regulation of sporulation and/or the maintenance of dormancy. Antibiotics produced by the aerobic sporeformers are often, but not always, polypeptides. Known antibiotic producers are Bacillus cereus (e.g. cerexin, zwittermicin), Bacillus circulans (e.g. circulin), Brevibacillus laterosporus (e.g. laterosporin), Bacillus licheniformis (e.g. bacitracin), Paenibacillus polymyxa (e.g. polymyxin, colistin), Bacillus pumilus (e.g. pumulin) and Bacillus subtilis (e.g. polymyxin, difficidin, subtilin, mycobacillin).

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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 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 121C 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 80C 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. all the tips in to the tip box. Then we wrap the tip box by Paper.
27

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

 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 121C 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 121C 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.
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SAMPLE COLLECTION:5 samples were collected from different region of Agra.

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

Area of Collection
Shastripuram , Agra Khandari , Agra Shahganj ,Agra Kargil, Agra Sikandra ,Agra

Type of Sample
milk milk milk milk milk

Type of Sample
R1 R2 R3 R4 R5

SAMPLE PREPARATION:
Taken 10 ml. Of milk in 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.

29

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 of animal 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

::::::-

PROCEDUREAll 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.
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PREPARATION OF BACILLUS DIFFERENTIAL MEDIA

Ingredients Yeast autolysate Mannitol Phosphate Potassium Magnesium Bromo cresol purple Agar Final pH (at 25C) gm/liter 0.20 5.00 1.00 0.20 0.20 0.0075 15.40 72

::::::::-

PROCEDURE 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. 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
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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. 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:

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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 Allow the slide to sit over the steaming water bath for 5 minutes, Remove blotting paper and rinse slide with water until water runs Flood slide with the counterstain safranin for 20 seconds and then View specimen under oil immersion lens with light microscope.

blotting paper with primary stain malachite green.

reapplying stain if it begins to dry out.

clear.

rinse.

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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 .

Procedure1.
2.

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

mixed with sterile loop. 3.

4.

bubbling. 5.

34

2.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.

Procedure
1.

Take 2-3 drops of (C6H4 [N (CH3)2]2.2HCl) oxidant on separate slides.

2. 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.

35

3. 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 37C for 24-48 hours. 3. After incubation, add five drops of sulfanilic acid and then five drops of alphanaphthylamine 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).
36

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.

37

4.HEMOLYSIS ON BLOOD AGAR

Hemolysis on blood agar is used for the preliminary or confirmatory identification of many types of clinically important bacteria. While it is factored into the differential diagnosis of a specific infectious agent, hemolysis type is not specific enough to be a final diagnosis criterion. The three hemolysis conditions continue to be described by terms that are somewhat confusing.

Alpha-hemolysis is a greenish discoloration of the blood agar surrounding a bacterial colony; it is a characteristic of Streptococcus pneumoniae. Beta-hemolysis indicates a zone of clearing in the blood agar in the area surrounding a bacterial colony. It is a characteristic of Streptococcus pyogenes, Bacillus cereus as well as some strains of Staphylococcus aureus. Gamma-hemolysis is actually a lack of hemolysis in the area surrounding a bacterial colony growing on blood agar. In fact, culture of bacteria on blood agar for the purpose of hemolysis classification is performed at 37oC in the presence of 5% CO2. This results in an overall brownish discoloration of the blood agar, from its original blood-red hue. An uninoculated blood agar plate (BAP) is shown on the left, above. Gamma-hemolysis would therefore describe bacterial growth that results in neither a greenish tinge to the discoloration (alpha-hemolysis) nor a clear zone that the observer "could read a newspaper through" (beta-hemolysis).

38

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). 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.

39

PROCEDURE:

1 or 2 loops full of microbial growth was scraped from culture media and suspended into 400 l 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.

40 l lysozyme was added in the vial, mixed well and incubate for 2 hours at 370C in shaking water bath. 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. The tube was centrifuged at 8000xg (10,000rpm) for 5 min. 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.

40

AGAROSE GEL ELECTROPHORESIS

Chemicals and Reagents: Tris Acetate EDTA Buffer(TAE Buffer) 50X :Tris base: Glacial Acetic Acid: EDTA: 242g 37.1ml 37.2g

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

41

 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: 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.

42

PCR (POLYMERASE CHAIN REACTION)

Reagent & chemicals
Distilled water 10x PCR buffer dNTPs 200 M primer(forward ) primer(Reversed) Taq DNA polymerase DNA sample :- 276.5 l :- 35.0 l :- 7.0 l :- 7.0 l :7.0l

:- 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. .

PROCEDURE43

1. 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.

RESULTS
44

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 Shastripuram , Agra Khandari , Agra Shahganj ,Agra Kargil, Agra Sikandra ,Agra Sample Code Type of Sample milk milk milk milk milk Colony Colour Grams Stain +ve +ve +ve +ve +ve Endospore Stain +ve +ve +ve +ve +ve

1. 2. 3. 4. 5.

R1 R2 R3 R4 R5

White+ Yellow White+ Yellow White White White

45

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

Table-2; Data of Biochemical Tests

46

S.No. Sample Code 1. 2. 3. 4. 5. R-1 R-2 R-3 R-4 R-5

Catalase Nitrate Test +ve +ve +ve +ve +ve Test +ve +ve +ve +ve +ve

Oxidase Test +ve +ve +ve +ve +ve

Blood Haemolysis Test +ve +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

47

2. OXIDASE TEST

3. NITRATE REDUCTION TEST

48

4. STARCH HYDROLYSING TEST

Hemolysis on Blood Agar

49

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)

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

50

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.

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
51

microorganism has been commercially exploited for over a decade. B. cereus has been used for industrial production of proteases, amylases, antibiotics, and specialty chemicals[63]. 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 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.

52

In addition, the availability of the complete genome sequence [64]and about 3,000 "y"-mutants constructed within the B. cereus Functional Analysis program [65] 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 [66]Many reports are available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and antiinflammatory properties of plants [67] 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. 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.

53

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