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INTRODUCTION THERMOPHILIC BACTERIA The Bacteria are a large group of unicellular, prokaryote, microorganisms. Typically, a few micrometers in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, waste, water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a milliliters of fresh water; in all, there are approximately five million (5×1030) bacteria on Earth, forming much of the world's biomass. A thermophile is an organism — a type of extremophile — that survive at relatively high temperatures, between 45 and 80 °C (113 and 176 °F). Many thermophiles are Archaebacteria. Thermophiles are found in various geothermally heated regions of the Earth such as hot springs and deep sea hydrothermal vents, as well as decaying plant matter such as peat bogs and compost. As a prerequisite for their survival, thermophiles contain enzymes that can function at high temperature. Some of these enzymes are used in molecular biology (for example, heat-stable DNA polymerases for PCR), and in washing agents. Thermophiles are classified into obligate and facultative thermophiles. Obligate thermophiles (also called extreme thermophiles) require such high temperatures for growth, whereas facultative thermophiles (also called moderate thermophiles) can thrive at high temperatures but also at lower temperatures (below 50 °C). Hyperthermophiles are particularly extreme thermophiles for which the optimal temperatures are above 80 °C. Thermophile is descended from the Greek: (thermotita), meaning heat, and Greek: (philia), love. Bacillus stearothermophilus The name Bacillus stearothermophilus (or Geobacillus stearothermophilus) is presumably intended to mean fat and heat loving. The most distinctive characters are the capacity to grow at 65°C. Bacillus strains capable of growing at temperature of 65°C and above do not belong to a single species, it is however a useful diagnostic character. Bacillus stearothermophilus occurs in soil. Bacillus is a large genus with many endospore producing species that are ever present. Some bacteria are not truly elongated, but can be kidney-shaped, curved (vibrios), or taper at each end (fusiform bacilli). These do not have the same arrangements as the cocci, but may be found in pairs (diplobacilli), or chains (streptobacilli). Bacilli may be single or adhere end to end to form chains. Some produce spores and some have flagella for locomotion. CLASSIFICATION OF Bacillus stearothermophilus Geobacillus stearothermophilus Scientific classification

Kingdom:	Bacteria Phylum:	Firmicutes Class:	Bacillus Order:	Bacillales Family:	Bacillaceae Genus:	Geobacillus Species:	Stearothermophilus Binomial name

Geobacillus stearothermophilus

MORPHOLOGY Bacteria of the genus Bacillus are Gram positive. Bacillus stearothermophilus are facultative anaerobic, endospore-forming rods. These organisms can vary in size from small filaments (0.5 x 1.2 µm) to large cells (2.5 x 10 µm). The formation of endospores is done in a way that is very similar to the genus Clostridium, an obligately anaerobic bacterium. Soils and manure are common habitats for Bacillus. Bacillus produces its endospores to protect itself from desiccation. Gram stain of Bacillus stearothermophilus with endospores The species of Bacillus is following: Bacillus species B. alcalophilus

B. alvei

B. amyloliquefaciens

B. aneurinolyticus

B. anthracis

B. aquaemaris

B. brevis

B. caldolyticus

B. centrosporus

B. cereus

B. coagulans

B. coagulans

B. firmus

B. flavothermus

B. fusiformis

B. globigii

B. infernus

B. larvae

B. laterosporus

B. lentus

B. licheniformis

B. megaterium

B. mesentericus

B. mucilaginosus

B. mycoides

B. natto

B. pantothenticus

B. polymyxa

B. pseudoanthracis

B. pumilus

B. schlegelii

B. sphaericus

B. sporothermodurans

B. stearothermophilus

B. subtilis

B. thermoglucosidasius

B. thuringiensis

HABITAT Thermophilic bacilli, including Geobacillus, are widely distributed and have been successfully isolated from all continents where geothermal areas occur. Geobacilli are also isolated from shallow marine hot springs and from deep-sea hydrothermal vents, with Maugeri et al. recently describing the isolation of three novel halo tolerant and thermophilic Geobacillus strains from high-temperature oilfields have also yielded strains of Geobacillus with reporting two novel species G. subterraneus and G. uzenensis. In addition Geobacillus species have also been recovered from artificial hot environments such as hot water pipelines, heat exchangers, waste treatment plants, burning coal refuse piles and bioremediation biopiles.

IMPORTANCE AND SIGNIFICANCE Initially more work has been concentrated upon the isolation of Geobacillus from natural and artificial high-temperature biotopes, but now attention has been focused upon strains of Geobacillus readily isolated from temperate soil environments. Initial studies surprisingly showed that thermophilic aerobic bacilli could be readily isolated in large numbers from a range of temperate soils. Subsequently this has been repeated, with similar findings, for a range of soils from geographically dispersed temperate regions. Industrial interest in Geobacillus species has arisen from their potential applications in biotechnological processes, for example as sources of various thermostable enzymes, such as proteases amylases lipases and pullanases Geobacillus species also have potential in generating products for industrial uses such as exopolysaccharides .In addition two strains of G. thermoleovorans have been described as producing large bacteriocins that exhibited a lytic activity on other strains of G. thermoleovorans and also a range of bacteria of medical importance including Salmonella typhimurium .A variety of potential environmental biotechnology applications involving Geobacillus species have been described, perhaps unsurprising given the seemingly ubiquitous capability of Geobacillus species to metabolize hydrocarbons. These days work is done to study novel applications for Geobacillus species, firstly in metabolizing herbicides and therefore being potential sources of genes for use in agricultural biotechnology, and secondly as having the ability to disrupt quorum sensing in certain Gram-negative bacteria. Organophosphonates, characterized by the presence of a carbon-to-phosphorus bond, are of widespread occurrence in the environment. Natural and synthetic organophosphonates are of importance, with the latter being extensively utilized in the chemical industry. The most important uses of synthetic Organophosphonates is as herbicides with Glyphosate, the world’s leading agrochemical,worth in excess of US$1 billion per year to its manufacturer, caldoxylosilyticus T20, isolated from domestic central heating system water, was found to be capable of metabolizing a range of organophosphonates including the herbicide glyphosate as sole phosphorus source. The organism is unique in that AMPA (aminomethylphosphonate) was released to the culture supernatant during growth upon glyphosate. In addition, in cell extracts prepared from G. caldoxylosilyticus T20 growing on glyphosate, a glyphosate-oxidoreductase-type activity, producing stoichiometric amounts of AMPA and glyoxylate, was detectable.While conversion of glyphosate to AMPA is the accepted mechanism for detoxification of this herbicide in soil no micro-organism that conclusively exhibits this capability has been isolated from soil. The gene encoding glyphosate oxidoreductase gox has been cloned from a strain of Ochrobactrum anthropi isolated from such a treatment facility, and used by Monsanto to genetically modify canola and other crop plants for enhanced herbicide resistance. Further analysis is required but it seems likely that thermophiles, such as Geobacillus, may provide an untapped resource of genes for use in agricultural biotechnology. The cell-density-dependent regulation of gene expression, or ‘quorum sensing’, is found in a plethora of bacterial species and plays a controlling role in a range of biological behavior and functions including virulence expression, conjugation, biofilm formation, bioluminescence and swarming. Although the target genes and regulatory mechanisms of quorum-sensing systems are extremely diverse, the general mechanism is highly conserved. Each bacterial cell produces a basal level of signal molecules that can move in and out of cells via diffusion or active transport .At low cell densities these pheromones are at low concentrations, and at high cell densities the signal molecules accumulate to a sufficient concentration that allows for the activation of cognate regulatory genes .Elucidation of the chemical nature of the signal molecules has revealed that Gram-negative bacterial quorum-sensing pheromones are mostly from the family of AHLs (N-acyl homoserine lactones), whereas, in Gram-positive bacteria the predominant signal molecules are peptide-based. Since signal molecule concentration is a major component of quorum-sensing controlled pathogenicity in many agricultural and medically important bacteria, it is seen as an ideal target for antimicrobial therapy. Several possibleways of interrupting the quorum-sensing circuitry have been reported .One possible strategy involves the inactivation of the signal molecules via enzymic degradation and is referred to as quorum quenching. Enzymes involved in this process either cleave the acyl side chain from the homoserine lactone component via an AHL-acylase or break open the lactone ring through the activity of an AHL-lactonase, encoded for by the gene aiiA The presence of lactonase activity has so far only been reported in a range of toxin-producing Bacillus species including B. cereus, B. thuringiensis and B. anthracis. Recent work has however shown that many Geobacillus isolates also possess the ability to degrade AHLs. In order to investigate the mechanism by which AHL disruption was mediated in these isolates, nucleotide primers specific for the aiiA gene in B. thuringiensis were used to screen each isolate. In one case,G. thermoleovorans, a PCR product of the expected size (900 bp) was obtained and upon sequencing of the product it was found that there was greater than 99% similarity to the aiiA gene described previously by Lee et al. At present it is unclear if mechanisms other than lactonase activity account for the ability of Geobacillus isolate to degrade AHLs; however, it is clear that the ability to quorum quench is much wider spread through Bacillus and related genera. Further studies are necessary to clarify if this metabolic capability has any environmental relevance for Geobacillus species, or indeed any other bacterium. Bacillus stearothermophilus is a common contaminant of dairy products, particularly milk powder. The organism is characterized by the ability of its spores to survive pasteurization (73 °C, 15 s) and grow at 65 °C. The organism is recognized as a problem in the manufacture of milk powder, as high levels of these bacteria may, after reconstitution of the milk powder, cause spoilage. The bacteria are present at low levels in raw milk, but may reach high levels in dairy products. This suggests that the bacteria grow during the manufacturing process. The growth of thermophilic bacilli during the manufacture of milk powder is believed to occur as a biofilm. Biofilms are defined as the growth of micro-organisms and their extracellular polymeric material on a surface. Micro-organisms in a biofilm are generally more resistant than planktonic cells to cleaning chemicals and sanitizers commonly used to clean food manufacturing plants. This creates a potential problem as incompletely cleaned manufacturing plant surfaces may enable rapid biofilm growth during the subsequent manufacturing run. Bacteria can be released from the biofilm contaminating product flowing through the plant. This is known as the biotransfer potential of the biofilm. The growth of vegetative cells of B. stearothermophilus is also on the surface of a milk powder manufacturing plant is also observed. The biofilms spores generally attach to surfaces at a greater rate than vegetative cells, a process facilitated by their relatively high hydrophobicity. Spores of B. stearothermophilus are of low hydrophobicity and attach to stainless steel surfaces much less efficiently than the spores of some other Bacillus species. The adherence, growth and release of several bacteria, including B. stearothermophilus, in a tubular heat exchanger. The doubling times of the cells in the biofilm and the potential for releasing significant numbers of bacteria into the liquid flowing over the biofilm is determined. The B. stearothermophilus from dairy manufacturing plants to attach to stainless steel surfaces is demonstrated by exposing stainless steel samples to suspensions of spores or vegetative cells and determining the numbers attaching using impedance microbiology. Spores attach more readily than vegetative cells. The attachment of cells to stainless steel was increased 10-100-fold by the presence of milk fouling the stainless steel. The growth of B. stearothermophilus as a biofilm on stainless steel surfaces is determined using a continuously flowing experimental reactor. The Thermophilic Bacillus species within the plant appears to be a likely cause of contamination of manufactured dairy products. Methods to control the formation of biofilms in dairy manufacturing plants are required to reduce the contamination of dairy products with thermophilic bacilli. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant. Decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes. Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might directly be determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis. Effect of acidification and oil is also studied on the thermal resistance of Bacillus stearothermophilus spores heated in food substrate. The effect of the addition of vinegar and/or oil to a food homogenate such tomato sauce, tuna and vegetables on the thermal resistance of Bacillus stearothermophilus spores. The results indicate that the food substrate without the addition of vinegar and oil and a pH value of 5.28 reduce the thermal resistance of B. stearothermophilus spores compared with that obtained in double-distilled water. The addition of vinegar reduced the pH of the substrate and consequently the D values were reducing. The addition of soya oil and vinegar to substrate until a pH of 4.81 further reduced the thermal resistance of the spores. The anticancer drug tamoxifen (TAM) is used as first line therapy in breast cancer. Although tamoxifen is usually considered an estrogen antagonist, several studies suggest alternative mechanisms of action. Bacillus stearothermophilus has been used as a model to clarify the antiproliferative action of tamoxifen putatively related with drug-membrane interaction. TAM induces perturbation of membrane structure along with impairment of bacterial growth. TAM inhibits bacterial growth and oxygen consumption of protoplasts as a function of concentration. Effects on oxygen consumption depend on the substrate used: NADH, allowing studying the full respiratory chain and ascorbate-TMPD to probe the final oxidase segment. The interaction of TAM with the respiratory components occurs at a level preceding the cytochrome oxidase segment. Genomic investigation of bacterial species important in medicine, biotechnology and the environment continues apace. The genome sequences of a number of Bacillus species have been completed (B. subtilis B. halodurans and B. anthracis recently reported the complete nucleotide sequence of the alkaliphilic and halotolerant Oceanobacillus iheyensis. This abundance of genomic information has allowed comparative genomic studies to be made between phylogenetically related bacterial species. Analysis suggests that the backbone of the genus Bacillus is composed of some 350 genes whereas specific sets of genes could be identified whose presence were essential for adaptation to extreme environments Although Geobacillus may be a recently described genus, considerable genomic information is available due to the ongoing genome sequencing projects of G. stearothermophilus and G. kaustophilus.The contribution to the understanding of Geobacillus with a genome sequencing project on G. thermoleovorans T80. Comparative genomic analysis will help to understand adaptations made within the thermophilic bacilli compared with related mesophilic strains. Rapid detection of Bacillus stearothermophilus using impedance-splitting has also been introduced. An impedance splitting method was used to detect Bacillus stearothermophilus in suspension and attached to stainless steel surfaces. The effects of bacterial metabolism on the impedance of the culture medium and the ionic layers of the measuring electrodes were recorded using the BacTrac 4000 microorganism growth analyser. Impedance changes were measured at 55o C. Seven of the eight media produce changes in the electrode impedance (E-value) and all media produced negligible changes in the impedance of the culture medium (M-value). Good correlations were obtained between cell numbers and the E-value measured over 18 h (r > 0.9) for the two strains of B. stearothermophilus tested in trypticase soy broth. The E-value correlations were used to estimate the numbers of both vegetative and spore forms of B. stearothermophilus as either planktonic or adhered cells. For the detection of B. stearothermophilus using impedance, only methods where the E-value impedance is recorded, can be used.

REVIEW OF LITERATURE Gram-positive, Aerobic or Facultative Endospore-forming Bacteria were identified by Cohn In 1872, and named the Bacillus subtilis. The organism is Gram-positive, capable of growth in the presence of oxygen, and forms a unique type of resting cell called an endospore. The organism represented what was to become a large and diverse genus of bacteria named Bacillus, in the Family Bacillaceae. Koch relied on Cohn's observations in his classic work (1876), The etiology of anthrax based on the life history of Bacillus anthracis, which provided the first proof that a specific microorganism could cause a specific disease. In 1876, Koch established by careful microscopy that the bacterium was always present in the blood of animals that died of anthrax. He took a small amount of blood from such an animal and injected it into a healthy mouse, which subsequently became diseased and died. He was able to recover the original anthrax organism from the dead mouse, demonstrating for the first time that a specific bacterium is the cause of a specific disease from this work Koch’s postulates were stated. Koch's postulates are: 1.	The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy animals. 2.	The microorganism must be isolated from a diseased organism and grown in pure culture. 3.	The cultured microorganism should cause disease when introduced into a healthy organism. 4.	The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent. The genus Bacillus remained intact until 2004, when it was split into several families and genera of endospore-forming bacteria, justifiable on the basis of ssRNA analysis. In order to accommodate former members of the genus Bacillus, the title has been changed to "Gram-positive aerobic or facultative endospore-forming bacteria". The unifying characteristic of these bacteria is that they are Gram-positive, form endospores, and grow in the presence of O2. The trivial name assigned to them is aerobic sporeformers. The ubiquity and diversity of these bacteria in nature, the unusual resistance of their endospores to chemical and physical agents, the developmental cycle of endospore formation, the production of antibiotics, the toxicity of their spores and protein crystals for many insects, and the pathogen Bacillus anthracis, have attracted ongoing interest in these bacteria since Cohn and Koch's discoveries in the 1870s. There is great diversity of physiology among the aerobic sporeformers, not surprising considering their recently-discovered phylogenetic diversity. Their collective features include degradation of most all substrates derived from plant and animal sources, including cellulose, starch, pectin, proteins, agar, hydrocarbons, and others; antibiotic production; nitrification; denitrification; nitrogen fixation; facultative lithotrophy; autotrophy; acidophily; alkaliphily; psychrophily; thermophily; and parasitism. Endospore formation, universally found in the group, is thought to be a strategy for survival in the soil environment, wherein these bacteria predominate. Aerial distribution of the dormant spores probably explains the occurrence of aerobic sporeformers in most habitats examined. R N McElhaney and K A Souza in 1972 worked on relationship between environmental temperature, cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus. A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes. Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might directly be determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis. Bacillus stearothermophilus disc assay for detection of inhibitors in milk was conducted by J W Messer, J E Leslie, G A Houghtby, J T Peeler, J E Barnett in 1976. A 2-part (A and B) collaborative study was conducted on a Bacillus stearothermophilus paper disc (12.7 mm) method to detect residual inhibitors in milk. The 18 participating collaborators assayed raw milk samples spiked with a beta-lactam (penicillin G). Of the 18 collaborators, 14 participated in part A and 16 in part B. Part A demonstrated that either Antibiotic Medium No. 4 or PM Indicator Agar is suitable for use in the assay. The lowest concentration detectable, not significantly different from 100% at the alpha = 0.05 level, was 0.008 unit/mL with either medium. Part B demonstrated that the sensitivity of the method is equal to that of the current AOAC method (16.131-16.136). The concentration of beta-lactam detected by 50% of the analysts was 0.003-0.005 unit/mL in this study, compared with 0.005 unit/mL reported in an earlier collaborative study on the current AOAC method. No false positive results were reported in part A or part B. All samples found positive by the confirmatory test in part B were correctly identified as a beta-lactam with commercial Penase discs. The lowest concentration detectable by the method, not significantly different from 100% at the alpha = 0.05 level, was 0.008 unit/mL. The method was adopted official first action. Several investigations emphasize the importance of membrane stability  in  relation  to heat  tolerance  ; Brock  (1967) has  suggested that  the molecular mechanism  of  thermophily  is possibly more related to the function and stability of the membrane than  to any special macromolecule. The capacity of thermophiles  to control  the physical properties  of  the cytoplasmic membrane  by changing its lipid composition in response to temperature changes has been supported by  the work  of  several  authors  (Daron,  1970; Ray  et  al.,  1971a, b; Weerkamp  & Heinen,  1972; Hasegawa et  al.,  1980). Ions, especially divalent cations  also have  a  role  in  thermostability (Ljunger, 1970; Stahl & Ljunger,  1976; Stahl,  1978; Mosley et al.,  1976). Ljunger (1970) has proposed  that  Ca2+,  accumulated  inside  the  cell  by  active  transport,  is  required  for  the thermostability of cell proteins. However, Mosley et al. (1 976) suggested that divalent cations increase membrane stability by  the formation of cation-bridges between the anionic groups of the acidic membrane phospholipids. Bacillus searothermophilus to know the morphology and habitat of B. stearothermophile. Thermophilic bacteria belonging to Bacillus genetic group 5 have been reclassiﬁed as being members of Geobacillus gen. nov., with G. stearothermophilus asthetypestrain. Geobacillus species, literally meaning earth or soil Bacillus, are widely distributed and readily isolated from natural and man-made thermophilic biotopes. As with many thermophiles there is considerable interest in potential industrial application of these bacteria and their gene products. This review describes two novel applications for Geobacillus isolates, ﬁrstly in the metabolism of the herbicide glyphosate and secondly in the metabolism of quorum-sensing signal molecules from Gram-positive bacteria. The genus Bacillus is a large, diverse collection of aerobic and facultatively anaerobic, rod-shaped Gram-positive bacteria that have undergone considerable reclassification as advances in molecular biology have revealed a high phylogenetic heterogeneity. Whereas Bacillus and related genera include psychrophiles, acidophiles, alkalophiles and halophilic bacteria able to utilize a wide range of carbon sources for heterotrophic growth or grow autotrophically, it is a specific group of thermophiles, the genus Geobacillus that this review concerns. Certain thermophilic aerobic spore-forming bacteria with growth optima in the range 45–>70°C were known to be classified into the genera Alicyclobacillus, Brevibacillus, Aneurinibacillus, Sulfobacillus, Thermoactinomyces and Thermobacillus. Molecular analysis, however, showed that the majority of such thermophilic bacteria described in the literature belonged to the genus Bacillus genetic groups 1 and 5. Subsequently group 5 isolates were found to be a phenotypically and phylogenetically coherent group of thermophilic bacilli with a high 16 S rRNA sequence similarity (98.5–99.2%). As a consequence in 2001 the thermophilic bacteria belonging to Bacillus genetic group 5 were reclassified as being members of Geobacillus gen. nov., meaning earth or soil Bacillus, with the well-known Geobacillus (Bacillus) stearothermophilus being assigned as the type strain. S Al-Awadi, M Afzal, S Oommen in 1978 influenced the biotransformation of testosterone to study the impact of chemical enhancers. Application of crude cell concentrates to produce Bacillus stearothermophilus-mediated bioconversion of testosterone at 65 degrees C for 72 h has been examined. After incubation, the xenobiotic substrate was added to the concentrated whole cell suspensions. The enhancer molecules were included in the whole cell suspension. The resultant products, after extraction into an organic solvent, were purified by thin layer chromatography and identification was carried out through spectroscopic data. Five steroid metabolites9,10-seco-4-androstene-3,9,17-trione,5alpha-androstan-3,6,17-trione,17beta-hydroxy-5alpha-androstan-3,6-dione, 3beta,17beta-dihydroxyandrost-4-ene-6-one and 17beta-hydroxyandrost-4,6-diene-3-one were identified as biotransformation products of testosterone. A possible biosynthetic route for these bioconversion products is postulated. Comparative studies of S-layer proteins of Bacillus stearothermophilus strains expressed during growth in continuous culture under oxygen-limited and non-oxygen-limited conditions has been carried out. M Sára, U B Sleytr University  Bodenkultur, Wien, Austria in 1982 described the specific properties of S-layer proteins from three different Bacillus stearothermophilus strains revealing oblique, square, or hexagonal lattice symmetry which was preserved during growth in continuous culture on complex medium only under oxygen-limited conditions in which glucose was used as the sole carbon source. When oxygen limitation was relieved, amino acids became metabolized, cell density increased, and different S-layer proteins from wild-type strains became rapidly replaced by a new common type of S-layer protein with an apparent subunit molecular weight of 97,000 which assembled into an identical oblique (p2) lattice type. During switching from wild-type strains to variants, patches of the S-layer lattices characteristics for wild-type strains, granular regions, and areas with oblique lattice symmetry could be observed on the surface of individual cells from all organisms. The granular regions apparently consisted of mixtures of the S-layer proteins from the wild-type strains and the newly synthesized p2 S-layer proteins from the variants. S-layer proteins from wild-type strains possessed identical N-terminal regions but led to quite different cleavage products upon peptide mapping, indicating that they are encoded by different genes. Chemical analysis including N-terminal sequencing and peptide mapping showed that the oblique S-layer lattices synthesized under increased oxygen supply were composed of identical protein species.

MATERIAL AND METHODS (A)	COLLECTION OF SAMPLE Care should be taken during collection of sample. The sample which are collected for culturing are not be collected from desert place. The soil samples for isolation of bacteria should be taken from crop land or nurseries in which less amount of fertilizer are used. For this project the 1st sample of soil is collected from lawn of CT Institute of Pharmaceutical Science Shahpur (Jalandhar) and 2nd sample was taken from the pot of CTIEMT Shahpur (Jalandhar). The sample were taken with clean and sterile spatula in a sterile petri plate. (B)	SAMPLE PREPARATION The two sample mixed properly in a petri plate and incubated at 80oC and then 65oC for 10 minutes. Weighed the three samples 10g each from incubated soil for culturing. (C)	CULTURING PROCEDURE Nutrient broth was prepared with following composition: COMPOSITION OF NUTRIENT BROTH Peptone			5g Beef extract		3g Sodium chloride		8g Distilled water		1000ml pH				6.5 i.	The flasks used to prepare NB were properly washed and sterlize by autoclaving. ii. Dissolved all solid ingredients in half volume of distilled water in flask and then made final volume by adding rest half of distilled water. iii. Plugged the flask properly and autoclaved at 121oC or 15psi for 20 minute. iv. After autoclaving tranferred 90ml of NB in each of three flasks aseptically. v.	10g of each sample was poured in to flasks containing 90ml of NB. vi. Incubated the flasks at 65oC for 48 hrs. Note the observation after 48 hrs. vii. Prepared serial dilution from each flask. viii. Poured 90ml of freshly prepared NB in 9 test tubes. Take 1ml of sample from the 1st flask and poured it into 1st test tube and serial dilution10-1,10-2,……….10-9. ix. Repeat the same procedure for 2nd and 3rd flask. x.	Icubated the serial dilution 65oC for 48 hrs. xi. Noted the observation after 48 hrs. xii. After this made further dilution of the 1st dilution for two times and note the observation.

SAMPLES Soil sample collected from lawn and pot from CT Group of Institutions

Soil sample after 48 hrs incubation in Nutrient Broth SERIAL DILUTION Serial dilution of sample After 48 hrs incubation of serial dilution (D)	PLATING (I)	 MEDIA PREPARATION COMPOSITION OF NUTRIENT AGAR Peptone			5g Beef extract		3g Sodium chloride		8g Agar			15g Distilled water		1000ml pH				6.5 i.	Nutrient agar sterilized by autoclaving a or 1210C 15psi for 20 minutes. ii. Poured approximately 2.5 of NA into sterilized petri dish aseptically. iii. After pouring ,the media was kept for solidification. iv. 1ml of sample from serially diluted tets tube having best microbial growth was spread, on to petri plate. v.	The petri plate were labeled carefully with date, type and dilution. vi. Then, petri plate were incubated at 650C (which supports thermophillic growth) for 48hrs along with beaker of distilled water to prevent excessive drying of NA plate. vii. After spreading sample, observation were noted and the process was repeated twice to obtain pure culture. viii. Then, culture were T-streaked (on to freshly prepared labeled NA plate) to isolate a pure culture. (II)	STREAKING Instruction before begin streaking procedure: i.	Wash hand and put on gloves and goggles. ii. Obtain all material and return to station. iii. Prepare work area. iv. Label agar plates with name of students, bacteria and date. v.	Begin streaking divide the plate in 1,2,3 &4 quadrant. vi. To avoid contamination, use a different loop when streaking each plate. vii. Place in 370C incubater upside down for 24hrs. viii. Clean reusable equipment and return to proper storage; put disposable in biohazards container. ix. Clean work area with surface disinfectant. x.	Remove gloves and wash hand with disinfectant. xi. Record results.

PROCEDURE i.	Hold the inoculating loop handle in one hand and the agar plate in the opposite hand. ii. Open the lid of the agar plate enough to insert the swab and spread the inoculum over  the surface of one quadrants of the agar plate. Do not streak over previously streaked lines. iii. Flame and cool the inoculating loop. iv. Turn the plate so that section two is in position. Streak the second quadrant by touching the loop into the first quadrant and streaking all the way across the second quadrant, making six to eight strokes. v.	 Flame and cool the loop. vi. Streak the third quadrant by touching the loop into the second quadrant and streaking into the third quadrant, making six to eight strokes. vii. Flame and cool the loop. viii. Streak the fourth quadrant in a manner to produce isolate colonies. Touch the loop to third quadrant and spread the organism into the fourth quadrant using a continuous streak in a “tornado” pattern. ix. Decrease the width of the streak horizontally and increase the distance between the streaks vertically. (Division of plate)	                                (Streaking on plate)

SPREADING After 48 hrs incubation of spreaded petri plate.

T-SREAKING After 48 hrs incubation of T-streaking. (E) FLOW CHART Took two soil samples from local nurseries.

Pack this sample in bag and label date and location of soil sample.

Mix soil sample in jar and incubate at 80°C for 10 minute.

Took three 10g soil sample from an incubated soil and pour under aseptically condition in three flasks containing 90 ml of Nutrient Broth.

Incubate this flask at 65°C for 48 hrs.

After 48 hrs dilute the sample and incubate at 65°C for 48 hrs repeat the process twice.

Spread the sample from test tube showing best growth on Nutrient agar plate.

Incubate NA plate at 65°C for 48 hrs and note the observation.

Took loop full of inoculums from these plates and T-streak a loop full sample on fresh NA plate.

Incubate the plate at 65oC for 48 hrs.

Note the observation after 48 hrs a whitish-cream color colony are observing on NA plate. Store the sample for further work. (F) STAINING Gram’s staining The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 –1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiella pneumoniae bacteria in lung tissue. It is a differential staining method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This reaction divides the eubacteria into two fundamental groups according to their stain ability and is one of the basic foundations on which bacterial identification is built. Gram staining is not used to classify archaea, since these microorganisms give very variable responses. Gram staining consists of four components 	Primary stain (Crystal violet, methyl violet or Gentian violet) 	Mordant (Gram's Iodine) 	Decolourizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture) 	Counter stain (Dilute carbol fuchsin, safranin or neutral red) PROCEDURE i.	Took smear on a glass slide and covered with few drops of one of the primary stains. Gentian violet is a mixture of methyl violet and crystal violet. The primary stain renders all the bacteria uniformly violet. ii. After a minute of exposure to the staining solution, the slide is washed in water. iii. The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant. iv. The slide is again washed in water and then decolorized in absolute ethyl alcohol or acetone. v.	 A mixture of acetone-ethyl alcohol (1:1) can also be used for decolorization. The process of decolorization is fairly quick and should not exceed 30 seconds for thin smears. Acetone is a potent decolorizer and when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and acetone acts more slowly than pure acetone. Decolorization is the most crucial part of Gram staining and errors can occur here. Prolonged decolorization can lead to over-decolorized smear and a very short decolorization period may lead to under-decolorized smear. vi. After the smear is decolorized, it is washed in water without any delay. vii. The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or saffranin. (G) BIOCHEMICAL CHRACTERIZATIONS TEST (1) Gelatin liquefaction test This test is used for identification of organisms, such as genus of Bacillus like Pseudomonas and Vibrio cholera. PRINCIPLE Gelatinase enzyme is secreted by the organisms such as Bacillus stearothermophilus which act on gelatin( liquefied ) and it is confirmed by placing the test tube at 4oc for 30 minute. REQUIREMENTS NUTRIENT GELATIN MEDIUM COMPOSITION Peptone		2.0g Beef extract		0.6g Gelatin			24.0g Distilled water		100ml

i.	Dissolve the solid ingredient in 100ml distilled water by heating. ii. Dispense 7ml in each test tube. iii. Sterilize by autoclaving at 121oc or 15psi for 15 minute. PROCEDURE i.	Inoculate the medium with test organism. ii. Incubate at 35-37oc for 72 hrs. iii. Chill the tube at 4oc for 30 minute. iv. Observe for positive gelatin liquefaction OBSERVATION Medium in fluid state		+ve test Medium in solid state		-ve test (2) CATALASE TEST This test is used to differentiate catalase producing Bacteria such as Bacillus, Staphylococci from noncatalase producing bacteria such as streptococci. PRINCIPLE Catalase produced by the organisms acts on hydrogen peroxide to produce water and oxygen (it is indicated by bubble) REQUIREMENTS i.	3% (V/V) Hydrogen peroxide. ii. Test tube.

PROCEDURE i.	Pour 2-3 ml of Hydrogen peroxide solution in a test tube. ii. Immerse growth of organisms in the test tube solution by using sterile wooden stick (or a sterile glass rod). iii. Look for immediate bubbling. OBSERVATION i.	Appearance of bubbles: Presence of catalase producing organisms ii. No, formation of bubbles Presence of non-catalase producing organisms. (3) METHYL RED TEST This test is performed to differentiate Enterobacteria. PRINCIPLE Some Enterobacteria when cultured in a Buffered glucose peptone water, ferment glucose to produce sufficient acidity, which gives red color with methyl red indicator (pH range 4.4-6.2). Color changes from red to yellow. PROCEDURE i.	Incubate a colony of test organisms into 0.5ml of sterile glucose phosphate broth. ii. Incubate overnight at 35 to 37oc. iii. Add a drop of methyl red indicator and observe the color. OBSERVATION Bright red colour		+ve test Yellow orange colour		-ve test

(H) ANTIBACTERIAL SENSTIVITY TEST INTRODUCTION AND IMPORTANCE OF PLANTS Woodfordia fructicosa Woodfordia fruticosa Kurz belongs to the family Lythraceae, which also includes other medicinal plants like Ammania baccifera, Lawsonia inermis, Lagerstroemia indica. The English names that are most frequently used for the plant are Fire flame bush and Shiranjitea. The plant is abundantly present throughout India, ascending up to an altitude of about 1500 m, and also in the majority of the countries of South East and Far East Asia like Malaysia, Indonesia, Sri Lanka, China, Japan and Pakistan as well as Tropical Africa. The local and traditional names are innumerable, especially in India, because of the widespread traditional use. The original Sanskrit name Agnijwala or Tamra-pushpi appears to be derived from the bright red colour of the flower and bark. In India, a few popularly known names are Dhataki, Dhawi, Jargi, Dhai, Harwari, Phulsatti, Dhavdi, etc. The full-grown leafy of shrub is about 3.5 m high, having long and spreading branches with fluted stems. The bark, characteristically cinnamon brown coloured and smooth, peels off in fibres and the young shoots are terete, often clothed with fine white pubescence. The leaves are opposite or sub-opposite in nature. Flowers are brilliant red, innumerable, the calyx is long, striated, covered with glandular dots, with a small campanulate base and a long slightly curved bright red tube that contracts above the included capsule. The petals are slightly longer than the calyx-teeth, narrowly linear, extended at the apex to a long fine point. The fruit are small capsules and membranous. The seeds are brown, numerous, minute, smooth, shining, angular and obovat. The plant is a well known non-wood forest produce that has long been in use regular demand amongst practitioners of traditional medicines in different South East Asian countries. In India, it is much used medicinal plant in Ayurvedic and Unani systems of medicines. Although all parts of this plant posses valuable medicinal properties, there is heavy demand for the flowers, both in domestic and international markets specialized in the preparation of herbal medicines. According to the Indian Systems of medicine, this flower is pungent, acrid, cooling, toxic, alexiteric, uterine sedative and anthelmintic, and is useful in thirst, dysentery, leprosy, erysipelas, blood diseases, leucorrhea, menorrhagia and toothache. It is considered as ‘Kapha’ (mucilage type body secretion) and ‘Pitta’ (energy dependant body activity) suppressant in the Ayurvedic concepts of medicine. Many marketed drugs comprise flowers, fruits, leaves and buds mixed with pedicals and thinner twigs of the plant. The flowers are being used in the preparation of Ayurvedic fermented drugs called ‘Aristhas’ and ‘Asavas’, and very popular in the Indian sub-continent and also in other South Asian countries. A popular crude drug (called ‘Sidowaya’ or ‘Sidawayah’) of Indonesia and Malaysia chiefly contains dried flowers of Woodfordia fruticosa. It has been used as an astringent to treat dysentery and sprue, and also for the treatment of bowel compliant, rheumatism, dysuria and hematuria in many South East Asian countries. It is also an ingredient of a preparation used to make barren women fertile. Tribal people in Chhatisgarh district of central India randomly use fresh flowers to stop bleeding in emergency cuts, while they prefer to employ dried flower powder to heal wounds more efficiently. Oil based flower extract has always been recommended for open wounds. Water decoction of the fresh flowers, either alone or in combination with ginger (Zingiber officinalle) or intrajua (Wrightia tinctoria), is used for the treatment of dysentery. Oral use of powdered bark in managing diarrhoea is well known. Successful treatment of otorrhoea by dried powdered flowers in tribal areas of Chhatisgarh is reported to be popular. Management of female specific disorders like leucorrhea and  dysmenorrhoea with flower based preparations is very popular among these tribes. An herbal composition containing Woodfordia fruticosa has been patented for the management of gynaecological disorders; it claims to prevent and treat anaemia due to excessive bleeding associated with menstrual disorders. The leaves of Woodfordia fruticosa are used as a folk medicine in India and Nepal. In case of fever, decoction of the Dhawi leaves (a popular name of the plant in this region) in combination with sugar and dried ginger is recommended. Terminalia arjuna The Terminalia arjuna belonging to family combritceae; is commonly in Indian peninsula. It is grown by the side of steams and very common in chotta- Nagpur region. The arjun tree Terminalia arjuna (Roxb.) is a well-known medicinal plant whose bark is extensively used in ayurvedic medicine, particularly as cardiac tonic. The bark is also prescribed in biliousness and sores and as an antidote to poison, and it is believed to have an ability to cure hepatic, congenital, venereal and  viral diseases. A decoction of its bark with cane sugar and boiled cow’s milk is highly recommended for endocarditis, pericarditis and angina1. Infectious endocarditis is an inflammatory disease of the endocardium, the internal lining of the human heart caused by bacteria such as staphylococci and gonococci. Among staphylococci, Staphylococcus epidermidis is one of the major etiological agents of this disease. The infections occur mainly in patients with prosthetic heart valves and during simple hospital procedures like catheterization, insertion of intra-uterine contraceptive devices, intravenous injections, etc. In our screening programme aimed at detecting biomolecules from plant sources, which can specifically act against S. epidermidis, we found that the bark extractsof T. arjuna possessed antibacterial activity. Bioactivity-directed fractionation of the active extracts yielded three known oleane compounds: arjunic acid (1), arjungenin (2), and arjunetin (3), which were found to possess activity against S. epidermidis. Arjuna bark is cardiolonic. It is also styptic, febrifugaland antidysentric. It posses diurelic and donic properties. The drug exhibits hypotensive action with vasodilation and decreased heart rate. ISOLATION OF EXTRACT For isolation of extract the leaves of woodfordia fructicosa plant are used which are obtained from Chohal, Hoshiarpur India, and Termminalia arjuna from the village Shahpur Jalandhar. The leaves are shade dried for about 20 days. About 100g dried leaves are grinded. The powedered leaves are extracted in soxhlet apparatus using different solvent i.e. Hexane, Acetone, Methanol. The solvents are then distilled off remaining the crude extract. DISK PREPARATION i.	Took a whatman filter paper. ii. Draw 30 circle with size 5mm on filter paper. iii. Cut the circle with sterile scissors under aseptic condition without touching internal disk space. iv. Wrapped the disk in Petri plate. v.	Sterilized the Petri plate by autoclaving at 121oC for 15 minute.

MEDIA PREPARATION Prepare 100ml media for antibacterial sensitivity with composition:- Peptone                    0.5g Beef extract              0.3g Sodium chloride       0.8g Agar                          3.0g Distilled water          100ml pH                               6.5 i.	Dissolve all solid ingredients in half of the volume and then add final volume of distilled water. ii. Plugged the flask. iii. Autoclaved the flask at 121oC or 15psi for 20 minute. POURING AND SPREADING i.	Took 6 Petri plate. ii. Washed with the detergent. iii. Set the hot air oven and dry the moisture of Petri plates. iv. Wrapped Petri plates with aluminum foil paper. v.	Autoclaved wrapped Petri plate at 121oC for 20 minute. vi. Pour the Approximately 25ml of NA in Petri plate. vii. Left plates for medium solidification. viii. Spreaded 20 micro liter of sample on NA plates aseptically.

PROCEDURE OF ANTIBACTERIAL SENSTIVITY TEST i.	Took spreaded petri plates. ii. Divided the plates into three quadrants. iii. Took steriled disk of whatman filter paper aseptically. iv. Pick up disk with sterilized forcep and dip in extract of different solvent. v.	Placed disk in 1st quadrant repeat the disk process using different extract for different quadrants. vi. Label the quadrants according to the type of extract in which the solvent was placed. vii. Incubated the plates Bacillus at 65oC for 24 hrs. viii. After 24 hrs measured zone of inhibition.

ANTIBACTERIAL SENSITIVITY TEST Plant extract

Disk of plant extract on petri plate

Before incubation of petri plates

RESULT AND DISCUSSION NUMBER OF COLONY

S.NO	SAMPLE	After24 Hrs	After 48 Hrs	After 72 Hrs 1.	I	20	37	53 2.	II	23	42	60 3.	II	21	39	57 The different numbers of colony are count after different incubation period 1.	Shape of colony The colony of Bacillus stearothermophilus is medium-sized, round type. 2.	Color of colony The colony of the organism is whitish-cream colored on agar plate. 3.	Consistency Due to motility, some colonies were spread-out on the plate with fingerlike projections. GRAM STAINING After gram staining performance rod shaped stained bacteria and non stained endospore are observe under light microscope. BIOCHEMICAL CHRACTERISATION TEST The different biochemical test are performed on Bacillus stearothermophilus sample I, II and III with dilution 10-3,10-4,10-5 result is following:- SAMPLE (I) S.No. B.C. TEST	DILUTION	OBSERVATION 1.	Gelatin liquefaction Test	10-3,10-4,10-5	Positive 2.	Catalase Test	10-3,10-4,10-5	Positive 3.	Methyl Red Test	10-3,10-4,10-5	Negative SAMPLE (II) S.No. B.C. TEST	DILUTION	OBSERVATION 1.	Gelatin liquefaction Test	10-3,10-4,10-5	Positive 2.	Catalase Test	10-3,10-4,10-5	Positive 3.	Methyl Red Test	10-3,10-4,10-5	Negative SAMPLE (III) S.No. B.C. TEST	DILUTION	OBSERVATION 1.	Gelatin liquefaction Test	10-3,10-4,10-5	Positive 2.	Catalase Test	10-3,10-4,10-5	Positive 3.	Methyl Red Test	10-3,10-4,10-5	Negative

1.	Gelatin liquefaction test The gelatin medium is present in fluid state after 72 hrs incubation and 30 minute chilling at 4oC the result is positive. 2.	Catalase test After inoculation of Bacillus stearothermophilus in 3% (v/v) and 3ml of hydrogen peroxide the bubble is formed in test tube the result is positive.

3.	Methyl red test After inoculation of Bacillus stearothermophilus in 0.5ml of phosphate broth and overnight incubation period at 37oC the 2-3 drop of methyl red indicator adding the yellow and orange colour are appear in test tube the test is negative. ANTIBACTERIAL SENSITIVITY TEST The antibacterial sensitivity of plant extract (Terminalia arjna and Woodfordia fruticosa ) prepared in different solvents- Hexane, Methane and Acetone for Bacillus stearothermophilus is as under: ZONE OF INHIBITION S.No. Sample	A. Hexane	A. Methanol	A.Acetone	W.f.Methanol	W.f. Acetone	W.f. Hexane 1.	Bacillus I	1.5cm	1.2cm	1.4cm	1.6cm	1.6cm	2.8cm 2.	Bacillus II	1.3cm	1.4cm	1.4cm	2.0cm	1.6cm	1.8cm 3.	Bacillus III	1.4cm	1.8cm	1.6cm	1.2cm	1.6cm	1.4cm

The antibacterial sensitivity of different solvent (hexane, Methane, acetone) extract of Terminalia arjuna and Woodfordia fruticosa are used. The maximum zone of inhibition is found above table its mean the Bacillus stearothermophilus is more antibiotic resistant bacteria.

COLONY OF Bacillus stearothermophilus Numbers, shape, colours, and consistency of colony GRAM STAINING OF Bacillus stearothermophilus Positive gram staining of Bacillus stearothermophilus. BIOCHEMICAL CHRACTERIZATION TEST Gelatin liquefaction test After inoculation of Bacillus stearothermophilus and incubation at 37oC for 72 hrs and chilling at 4oC for 30 minute gelatin media in fluid state. Catalase test Bubble formation after incubation colony of Bacillus stearothermophilus in 3% H2O2. Methyl red test After incubation of Bacillus stearothermophilus in glucose phosphate broth at 37oC for 12 hrs and adding 2-3 drop of methyl red indicator yellow colour are appear. ANTIBACTERIAL SENSITIVITY TEST Zone of ihibition after icubation Bacillus stearothermophilus with extract of Terminalia arjna and Woodfordia fruticosa which extracted using solvent Acetone, Methanol and Hexane. Number of colony of Bacillus stearothermophilus which are of medium-size, round and cream color are counted. Growth is observed at 65o C under aerobic conditions. Due to motility some colonies were spread-out on the plates with fingerlike projections. Gram stains of the isolated colony indicate that bacteria are gram-positive rod, some of which contained endospores found near the terminal end (subterminal) of the cell. The gelatin liquefaction test and catalase test ( i.e. confirmation test) is positive, its mean the gelatinase and catalase enzyme is secreted by  Bacillus stearothermophilus  and the methyl red confirmation test is negative mean  the sufficient acidity is not produced by Bacillus searothermophilus. Antibacterial sensitivity test shows that the zone of inhibition. It mean that Bacillus stearothermophilus is more antibiotic resistant bacteria due to its capacity for spore formation.

CONCLUSION

Due to the observance of rod-shaped bacteria with endospores, along with positive Gram stain and positive Gelatin liquefaction catalase and negative Methyl red tests, it was concluded that Bacillus stearothermophilus has been successfully isolated. From these results, it was determined that Gram-positive, aerobic rod-shaped bacteria had been isolated in pure culture. One addition that made in our procedures was to put the beaker with water in incubator. This is done to prevent over drying of the agar plates stored at 65 oC. In this protocol, the appearance of endospores within the rod-shaped cells under the microscope was a major indicator that Bacillus stearothermophilus was present. Endospores were located either towards the side of the cell or at the very end of the rod shaped cell. Not all cells formed endospores synchronously due to the fact endospore formation is instigated by a change in the environment (lack of nutrients, temperature change, desiccation, overgrowth) or age of the cell. Thus, endospore formation reflects conditions that are not optimal for the growth of the vegetative cell (cell without endospores). Therefore, some cells have endospores while others lack them. B. stearothermophilus isolation is supported by gelatin liquefaction and catalase test. This test was performed to ensure that a similar Bacillus species was not insolated instead B. stearothermophilus. Bacillus coagulans is similar in that it is a gram-positive thermophile, rod shaped and with endospore formation. The major difference is that Bacillus stearothermophilus gives positive test for liquefaction of gelatin and catalase while Bacillus coagulans doesn’t. Thus, even though Bacillus coagulans is rarely found in soils, the test is performed to ensure that there is no false isolation, or thermophilic contamination from laboratory sources such as from tools, incubator, air or other equipment. Also as Bacillus stearothermophilus is an obligate aerobe, a positive test for the presence of the enzyme catalase is indicated by the formation of oxygen bubbles. It would be interesting to observe which biomass Bacillus stearothermophilus is most efficient at decomposing (or which nutrients it can efficiently utilize). Bacillus stearothermophilus is commonly found in compost piles as a material degrader. However, what specific material Bacillus stearothermophilus most effectively degrades is not known. Thus, Bacillus stearothermophilus could be plated out on agar plates containing different biomass (dead plants/leaves, manure, common soil) and then the overall growth rate and nutrient utilization can be observed. Growth rate could be quantified by performing colony counts at successive time intervals (ex. 24hrs, 48hrs, 72hrs, etc.), while nutrient utilization could be observed by speed at which endospores are formed (assuming that endospores are formed by lack of nutrients). Therefore, it can be proposed that greater the growth rate and nutrient utilization of a certain biomass the more effective Bacillus stearothermophilus may be used in bioremediation of that specific compound.

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