Physicological Characterization of M.VITRJS5

Physicological Characterization of M.VITRJS5 Monica Samal, Teena Patra, Suthindhiran K Abstract Magnetospirillium sp. is a Gram – negative ÃŽ ±-proteobacterium which has the ability to move towards the geomagnetic field lines of Earth’s magnetic field. The group of bacteria is collectively called Magnetotactic bacteria (MTB). MTB produce intracellular organelles called magnetosomes which help them in its search for optimal living conditions in complex environment. MTB is a gradient – requiring microorganism that are difficult to replicate in growth medium in the laboratory. In this work the physiological characterization of MTB strain Magnetospirillium sp. VITRJS5 was done as well as a modified growth medium for the growth of Magnetospirillium sp. VITRJS5 was developed. The media components are studied based on the physiological characteristics of the bacteria. The role of electron donors, electron acceptors, nitrogen sources, carbon sources, iron containing compounds, phosphates, reducing agents, on the growth of the bacteria in the MS1 media was studied. The growth of the bacteria in the novel modified MS media was analysed and compared with commercial MTB media such as MSGM (Magnetospirillum growth media) and Schuler’s oxygen – sulphur gradient media. An increase in growth is observed in the modified MS media compared to the commercially available media. 1. INTRODUCTION: Magnetotactic bacteria are fastidious microorganisms that represent a morphologically and physiologically diverse group of bacteria. The presence of intracellular, membrane bound nano-sized magnetic particles called magnetosomes assists the bacteria in sustaining an optimal position in redox gradient habitats. Magnetosomes are crystals of iron mineral which consist of either iron oxide (Fe3O4) or iron sulphide (Fe3S4). A combination of aerotaxis, magnetotaxis and chemotaxis helps MTB to move away from oxygen in surface water, thus directing them in maintaining an optimal position in and around the oxic-anoxic interface (OAI). Magnetospirillum sp. is the most studied and well understood group of bacteria among MTB. Magnetospirillum sp. was mostly isolated from fresh water habitats and many pure cultures were available in many laboratories around the world. Magnetotactic Bacteria (MTB) species have characteristic organelles called magnetosomes that contain magnetic crystals. These magnetosomes give them the ability to move in response to the earth’s magnetic field (magnetotaxis). MTB were first observed by Salvatore Bellini in the year 1963 and collected from different freshwater environments near Pavia, Italy [1,2]. The most studied species of Magnetospirillum genus are species of Ms. gryphiswaldense [5], Ms. Magneticum[7], Ms. magnetotacticum [8]. The MTB strains isolated from marine or brackish environments include: the coccoid strains: MO-1[9] , Magnetococcus marinus strain MC-1[10] and Magnetofaba australis (IT-1)[11] ; the vibrioid strain: Magnetovibrio blakemorei strain MV-1[12] and the spirilla Magnetospira thiophila (MMS-1)[13] and Magnetospira sp. strain QH-2[14] ; all of which belong to the Alphaproteobacteria. Most magnetotactic bacteria (MTB) are known to be phylogenetically similar to the Alphaproteobacteria, Gammap roteobacteria and Deltaproteobacteria classes of the Proteobacteria and the Nitrospirae phylum with response to magnetosome formation[6]. Magnetotactic bacteria are known to thrive in sediments or chemically stratified water columns, where they occur mostly at the oxic-anoxic interface (OAI), the anoxic regions of the habitat, or both [3]. Although the detection of these in samples is relatively simple [4], magnetotactic bacteria are a fastidious group of prokaryotes, and special culture conditions are necessary for their isolation and cultivation. There is clear evidence that the availability and diversity of MTB in different environments is majorly influenced by salinity and also, iron availability, temperature and concentrations of sulfur and nitrogen compounds are also seen to be clearly important in the distribution of MTB. The respiratory forms of metabolism of genus Magnetospirillum are either chemoorganoheterotropic, using organic acids and carbon source or electron donors. Mostly Magnetospirillum sp. capable of autotrophic and mixotrophic growth and utilize nitrate as alternative terminal oxygen acceptor. Magn etospirillum are obligate microaerophile that requires oxygen even when nitrate is present in the medium. The present study,examined the phylogenetic affiliations and physiological characteristics of Magnetospirillium sp. VITRJS5. To characterize physiological parameters, microaerophillic batch experiments were performed to determine the following: (1) growth pH and temperature ranges, (2) Optimum electronic donor/carbon source and electron acceptor. (3) optimum nitrogen and phosphate source (4) optimum Iron source (5) optimum reducing agent/ sulphur source. Once the preliminary screening of the compounds over different concentrations of the nutritional requirements is determined to develop a modified growth media. 2. Methods and materials: 2.1. Media and microorganism Magnetospirillum VITRJS5 (Accession number: KM289194) is a novel MTB isolated from the fresh water sediment. They are spiral in shape and move towards the earth’s magnetic field. They have polar flagellum at each end of the cell. The strain was obtained from Marine biotechnology and biomedicine lab, VIT University. The bacteria were sub-cultured in Magnetospirillum growth media (MSGM) under microaerophillic conditions Magnetospirillum VITRJS5 was initially grown in previously standardized minimal media MS1 to analyse the growth conditions in the presence of electron donors and acceptors. MS1 media per litre containing K2HPO4 – 850mg, Na2HPO4 – 850mg, NH4H2PO4 – 500mg, MgSO4 – 100mg, FeSO4 7H2O – 5mg, Na2MoO4 – 0.2mg, EDTA – 10mg and Mineral solution 10ml Electron donor and acceptor has to be added accordingly. From the earlier studies, the optimum temperature and pH for the growth of Magnetospirillum is 28 °C and 7.5 pH. 2.2 Determination of electron acceptors and concentration of selected acceptor: For this, 50 ml of MS-1 media was added in five serum bottles and 50 mg of electron donor which in this case is sodium acetate (CH3COONa) was added to each bottle. Then 50 mg of five different acceptors were added in different bottles. These five acceptors are nitrite, selenite, nitrate, thiosulphate and sulphate. Nitrogen was sparged to the media to make it anaerobic, closed with butyl rubber stopper and sealed with aluminium caps. All the anaerobic culturing was performed according to Hungate 1950[15]. The Magnetospirillum VITRJS5 was inoculated to MS1 media. These bottles were kept for overnight incubation in a shaker incubator. Now the different concentrations of selected acceptor is determined .It is done for concentration of 5mM, 10 mM , 15mM, 20 mM and 30 mM . Steps for anaerobic culture was repeated. The absorbance is checked in UV-VIS spectrophotometer at wavelength of 595 nm. 2.3. Determination of electron donors and concentration of selected donor: For this, 50 ml of MS-1 media was added in five serum bottles and 50 mg of electron acceptor which in this case is nitrate was added to each bottle. Then different donors were added in different bottles. These donors are methanol (250  µl), ethanol( 50 µl), glycerol (50 µl), pyruvate (82.5 mg/ 15mM) , citrate (221 mg/ 15mM),succinate (202.6 mg/15mM), lactate(110 µl/15mM), glucose (135.12 mg/15mM), sucrose (256.9 mg/15mM), Ferrous chloride (121.65 mg/15mM)and thiosulphate (186.1 mg/15mM).Nitrogen gas was bubbled into each bottle for anaerobic condition and sample VITRJS5 strain was inoculated in each bottle. These bottles were kept for overnight incubation in a shaker incubator. Narrowing down the number of donors based on the growth of sample. Different concentrations of selected donor are determined. Selected donors are added in different concentration of 5mM, 10mM, 15mM, 20mM and 30 mM. The steps for anaerobic culture are repeated. The absorbance is checked in UV-VIS spectr ophotometer at 595 nm. 2.4. Determination of nitrogen source for optimum growth: MS-1 Media was prepared without nitrogen source (MS-1 media without NH4H2PO4 ) and with the selected concentration of electron donor and acceptor. For this, activity of NH4H2PO4 was checked in different concentrations (5mM, 10mM, 15mM, 20mM and 30 mM) against different concentrations of NH4Cl(5mM, 10mM, 15mM, 20mM and 30 mM).The above described procedure of anaerobic growth was repeated. The absorbance was checked at a wavelength of 595 nm. 2. 5. Determination of reducing for optimum growth: MS-1 media with selected concentration of donor and acceptor was prepared. For this, activity of thiol glycolate was checked in different concentrations of 5mM, 10mM, 15mM, 20mM and 30 mM against cysteine-HCl 4% at concentrations of 50  µl , 100  µl, 150  µl, 200  µl and 500  µl. The procedure for anaerobic culture growth was repeated. The absorbance was checked at 595 nm. 2.6. Determination of iron source for optimum growth MS-1 media with selected concentration of donor and acceptor and without FeSO4 . 7H2O was prepared. For this, activity of ferrous sulphate was checked in different concentrations of 5mM, 10mM and 15mM against ferric citrate at concentrations of 0.5mM, 1.0mM and 1.5mM and against ferric quinate at concentrations of 0.5mM, 1.0mM and 1.5mM. The procedure for anaerobic culture growth was repeated. The absorbance was checked at 595 nm. 3. Result: 3.1 Electron acceptor and its concentration for optimum growth: On incubating the strain M.VITRJS5 in the MS1 media with the various electron acceptors, we obtained various results as seen in table1. From the table, we find that Nitrite, Nitrate and Selenite good electron acceptors for the strain. But we negate the role of Nitrite and Selenite as in nitrite, growth is observed after 2 weeks and selenite is considered toxic. Thus, Nitrate is considered as the most probable electron donor. Next, different concentrations of Nitrate are added to MS1 media to see which concentration of nitrate supports maximum growth (as seen in Table 2 and fig.1) Table 1. Tabular representation of growth with various electron acceptor. Table 2. Tabular representation of absorbance of different concentration of nitrate Fig.1 Graphical representation of the growth with different concentration of nitrate From the above table and fig.1, it is seen that 5mM of nitrate shows maximum growth on all three days. Thus, there is optimum growth seen in 5mM serum bottle. 3.2. Electron donor and its concentration for optimum growth On incubating the strain M.VITRJS5 in the MS1 media with the various electron  donors, we obtained various results as seen in table1.From the table 3, it is seen that growth of M.VITRJS5 is more pronounced with electron donors Ethanol, Glycerol, Pyruvate, Succinate, Lactate, Glucose and Sucrose along with Acetate which was already being used as an electron donor for other MTBs. Among these, Pyruvate, Sucrose and Acetate showed almost double the growth as seen in other electron donors. But we negate Sucrose as an electron donor as it is not economical. Later, different concentrations of Nitrate are added to MS1 media to see which concentration of nitrate supports maximum growth (as seen in Table 2 and fig.1) Table3. Representation of growth with various donors Table 4. Tabular Representation of absorbance of growth with different concentrations of pyruvate Fig 2. Graphical representation of absorbance of growth with different concentrations of pyruvate As seen in the above two graphs and tables, 30mM concentration of pyruvate shows highest growth in comparison with the other concentrations of acetate and pyruvate. 3. Nitrogen source and its concentration for optimum growth: The next step is incubation of M.VITRJS5 strain in MS-1 media(without NH4H2PO4) with Ammonium chloride and Ammonium hydrogen phosphate to determine the better nitrogen source. Fig 4. Graphical representation of absorbance of growth with different concentrations of NH4H2PO4 As seen in figure 4, 5 and tables 6,7, it is observed that the best growth is seen in bottles of Ammonium hydrogen phosphate(NH4H2PO4) rather than that of Ammonium chloride(NH4Cl). The most consistence growth is seen in 20 mM concentration of NH4H2PO4 as compared with other concentrations. 3.4. Carbon source: Since it has already been confirmed that pyruvate acts as the best electron donor, the same can act as a carbon source. 3.5. Phosphorus source: Ammonium hydrogen phosphate acts both as a nitrogen source as well as phosphorys source. 3.6. Sulphur source: Next the M.VITRJS5 strain was incubated in prepared MS-1 media with Sodium thioglycolate and cysteine HCl 4% to determine the better sulphur source. Fig 6. Graphical representation of absorbance of growth with different concentrations of Sodium thioglycolate As seen in fig 6,7 and table 8,9, 50 µl concentration of Cys –HCl shows highest growth as compared to the other concentrations. There was no growth in other concentrations due to the reducing properties of Cysteine HCl and Sodium thioglycolate. MTBs are microaerophilic and hence, would not grow in anaerobic condition. Sulphur is a reducing agent and hence, would remove oxygen species from the media. Therefore, there is less or minimal growth of the bacteria. 3.7 Iron source : Table 10. Tabular representation of absorbance of growth with different concentrations of Ferric citrate Fig 8. Graphical representation of absorbance of growth with different concentrations of Ferric citrate Table 11. Tabular representation of absorbance of growth with different concentrations of quinate Fig 9. Graphical representation of absorbance of growth with different concentrations of Ferric quinate 3.8 Comparison of new media with existing media: 4. Discussion: As already mentioned above, Magnetotactic bacteria (MTBs) are microaerophilic bacteria which have the ability to biomineralise membrane-encased, single-magnetic-domain mineral crystals (magnetosomes) and hence, cause the cell to orientate along the Earth’s geomagnetic field. These mostly occur in aquatic bodies ranging from saline to freshwater environment. Our basic objective in this study was to test the growth of the microaerophilic strain using different concentrations of electron donor, electron acceptor, nitrogen source, carbon source, phosphorus source, sulphur source, iron source, etc. From the study, the following observations have been noted down: The strain M.VITRJS5 was seen to grow well in 5mM concentration of Nitrate (electron acceptor), 30mM concentration of Pyruvate (electron donor and carbon source) and 20mM concentration of Ammonium hydrogen phosphate (nitrogen source and phosphorus source). Also, the growth of the strain was seen to be minimalized in case of Sodium thioglycolate and Cysteine HCl as these are strong reducing agents and thus, would reduce the amount of oxygen species in the media. As mentioned above, these bacteria are microaerophilic and hence, would not grow in sulphur sources (except in 15mM Sodium Thioglycolate and 50 µl Cysteine HCl). The comparison of the present media for Magnetospirillum growth media (MSGM) Schà ¼ler Magnetospirillum isolation media and the new prepared media was done. The new media contained sodium nitrate as acceptor, sodium pyruvate as electron donor and carbon source, ammonium hydrogen phosphate as nitrogen and phosphorus source, ferric citrate as iron source and cysteine HCl as sulphur source. Magnetotactic bacteria has the ability to produce magnetosomes which has stimulated and motivated a new field of research involving scientific and biomedical applications of MTB and diverse commercial which could be improved using magnetic nanocrystals. But the main drawbacks to the application of magnetosomes involves the fastidious nature of MTB related to the growth. Thus,it is difficult to culture them on a large scale, and it is important to understand genetic/environmental control for magnetosome synthesis.

What is a sample?

Whereas a population is the entire group of objects that a certain researcher is interested in; a sample is defined as the fixed number of objects you get from a certain population. For example, Amelia wants to know if red flowers attract bees more than yellow flowers. In order to prove this assumption, Amelia takes a sample of a red flower (e. g. rose) and a yellow flower (e. g. a sunflower). There are lots of flowers that are red and yellow in color. Amelia could not afford to obtain every red and yellow flower in order to prove her assumption.Thus, it is practical for her to take a â€Å"representative† from all the red flowers and a â€Å"representative† from all the yellow flowers. Taking â€Å"representatives† from the entire population, you could now call these â€Å"samples†. It is essential to remember that the fundamental assumption underlying most of the theory of sampling is random sampling. This consists of the selection of individuals from th e population in such a way that each individual of the population has an equal chance of being selected. The process of such selection is called random sampling.The aim of the theory of sampling is to get as much information as possible, ideally all the information about the population from which the sample has been drawn. From the parent population, in particular, we would like to estimate the parameters of the population or specify the limits or ranges within which the population parameters are expected to lie with a specified degree of confidence. At work, we use sampling to prove or test something. For example, you want to determine if the new time management scheme will be beneficial to cut the costs on your company.So, as a manager, you could take some employees to undergo this new time management scheme in order to see if the new process is suitable for both the company and the employees. 2. What are the differences between the binomial and normal distributions? What are the similarities between the binomial and normal distributions? The normal distribution is the most commonly encountered distribution range in science. Random variables in normal distribution should be capable of assuming any value on the real number line, though this requirement is often not applied.For example, height at a given age for a given gender in a given racial group is adequately described by a normal random variable even though heights must be positive. A continuous random variable X, taking all real values in the range. The graph of variables with normal distribution is a symmetrical, bell-shaped curve, centered at its expected mean value. Typically, a binomial random variable is the number of successions in a series of trials in binomial distributions.For example, the number of ‘heads' occurring when a coin is tossed 50 times; thus a discrete random variable X is said to follow a binomial distribution with parameters n and p. However, the probability trials must meet the following requirements: a. the total number of trials is fixed in advance; b. there are just two outcomes of each trial; success and failure; c. the outcomes of all the trials are statistically independent; d. all the trials have the same probability of success. The similarity of normal and binomial distributions rely on the use of random variables as part of the data and their values could be both positive and negative.3. What do confidence intervals represent? Give an example of the use of a confidence interval. Before a simple research question could be resolved like, for instance, â€Å"What is the mean number of flowers that one person can remember? † it is necessary to specify the population of people to which this question will be addressed. The researcher could be interested in, for example, children under the age of 12 and girls. For the present example, assume the researcher is interested in all girls aged 9. Once the population is specified, the next step is t o take a random sample from it.In this example, let's say that a sample of 10 girls is drawn and each student's memory tested. The way to estimate the mean of all girls would be to compute the mean of the 10 girls in the sample. Indeed, the sample mean is an unbiased estimate of ? , the population mean. However, it will certainly not be a perfect estimate. By chance it is bound to be at least either a little bit too high or a little bit too low. For the estimate of ? to be of value, one must have some idea of how precise it is. That is, how close to ? is the estimate likely to be?So we use the confidence intervals to determine how close would be the unbiased estimate we have in our sample to the values that is indicated in the population mean. If the number of flowers that the 10 girls remembered were: 4, 4, 5, 5, 5, 6, 6, 7, 8, 9 then the estimated value of ? would be 5. 9 and the 95% confidence interval would range from 4. 71 to 7. 09. The wider the interval, the more confident yo u are that it contains the parameter you are interested in. The 99% confidence interval is therefore wider than the 95% confidence interval and extends from 4. 19 to 7. 61.

Between Realism and Idealism

A great deal of conflict in the social life of individual people has been brought about by the existence of these concepts raising some questions of importance. Should an individual aim so high to an extent that is impossible to reach or aim average that is achievable?Should the individual assume on optimistic stand or a pessimistic one? Should the individual be good to others or fair to them? The existence of the various branches with regard to these categories proves that idealism or realism is never right with respect to all the branches.The issue of idealism and realism as a debate addresses the issues of whether an individual’s life as well as actions should be good as has been offered by the dogma of the Christians way of life. The issue of whether the individual should possess a caring sense as well as be fair with regard to the daily life is a determinant of whether the individual is living in a realistic or in an idealistic way of life.As for example, the issue of whe ther an individual an individual should drink or not depends on the stand the individual has taken with regard to the two extremes. It is evident that a person can drink and do no evil to the others while another person who does not drink at all may cause harm of significantly high magnitude to the life of others.It is a clear fact that each individual person is potentially capable for being good internally. In a realistic sense however some people are so static with regard to matters of change. As pertains the issue of whether the individual should drink or not, the goodness or the fairness of this will highly depend on the stand that the individual has taken.It is a possibility that one individual drinks and commits no harm to others while another person refraining from the activity may be of great trouble to other people. Furthermore the Christian Bible does not condemn drinking as an activity, but drunkardness as the effect of the activity (Tomuschat 83).As a clear guide as to t he best stand to take in life, a conservative approach to life is the most noble approach whereby the actions of the individual are determined by the need to be fair, obey the rule of justice as well as equality in a real life situation. It is good to make all the people get what is right for them, treating everybody in the same way and judging everybody the actions responsible for the person.The decision by the individual to arrange for a Sunday trip will also depend on the stand that the individual has taken since the trip may exhibit different motives. A trip that is meant for good as well as for the service of others is justified even at the context of the bible (Tomuschat 113).The contrasting belief is for people to live in a life of goodness, mercy as well as equality bearing in mind that every person bears intelligence which is a guide towards the actions taken, all of which are backed by some reason.The capacity for understanding is a clear lead to goodness. A fair treatment to all the people is important sign if the individual underwent a bad life in the childhood or is subjected to unconducive circumstances that fall beyond the control of the individual is a result of poverty wages, there occur a requirement for compensation so that everyone enjoys life in an equal capacity. The negligence of other people is responsible for the actions of others in the light of the considerations of the fallibility of people. Human beings are prone to making mistakes and this is so natural.A good person may be apprehended and aligned in the court of law for having committed a mistake. In the light of this argument, the possession of a Christian wife or otherwise similarly depends on the stand of the individual wife in as far as the two extremes of realism and idealism are concerned. The Christian wife is supposed to be a model for idealism, a stand which if defeated by realism, which bears potent influence to the behavior as well as the action of human, is equivalent with having a wife who is not a Christian (Tomuschat 213).ConclusionIn the consideration of the argument between justice and goodness, non is comparable to what God is in a position to handle which is usually in a perfect way and being neither rebel nor conservative and offering a question less situation as regards the correct balance in existence between the boundaries of justice on one side and grace on the other.In the case of the human being, who posses the characteristics of inferiority with respect to several aspects they can only grasp these concepts independently and one at every opportune time thus facilitating into parties of different orientations. It therefore important for an individual to lead a life of self awareness, that is instrumental towards the discovery of the stand of the individual with respect to idealism or realism.Work citedChristian, Tomuschat, Between Idealism & Realism. 2nd ed. USA: Oxford UniversityPress, 2004.  

Symbolism â€Rendering the Hidden Meaning of the Story

Symbolism –Rendering The Hidden Meaning of The Story Symbolism is an important element in reinforcing the meaning of a story. It is a kind of formalist strategies which helps the reader understand the images that the author is trying to say in words. Symbolism is widely used in â€Å"The Story of An Hour† and â€Å"The Cranes†, which use conventional symbols, literary symbols, and even allegory. â€Å"The Story of An Hour† is written by Kate Chopin. Mrs. Mallard hears of her husband’s death from her husband’s friend Richards. At first, she feels depressed, but then she feels free and can have her own new life after thinking things through. Sadly, her â€Å"new† life is cut short by the abrupt return of Mr. Mallard. Mrs. Mallard, who has a bad heart, was so†¦show more content†¦The couple is symbolized as crane but they commit suicide to end their dreadful life suffering from their sickness. The author uses the water and hull of car as symbols in the story. â€Å"the water looked like metal, still and hard.† (Meinke, 259) and â€Å"The hull of the car gleamed beetle-like –dull and somehow sinister in its metallic isolation.† (Meinke, 260) shows the symbolization by describing the water and hull of the car. Both of the descriptions are related to the gun that the couple uses for committing suicide. They are hard, metallic like, and callous which is just like the gun. These also symbolize the grim tone in the story. It is stillness because the couple is going to commit suicide. The final symbol in the story is the direction that the cranes fly to. â€Å"†¦their great wings beating the air and their long slender necks pointed like arrows toward the sun.† (Meinke, 260) After the couple kiss and close the eyes, the cranes fly toward the sun. This is a symbol of western paradise of ultimate bliss, which means they kill themselves by shooting the gun to end their lives. Flying toward the sun is a traditional symbol that says that people will go to paradise after they die. The combination of those symbols in the story raises the controversy of suicide to the readers. Refering to the story, the couple chooses euthanasia because they are in pain from sickness. It also raises the question of whether weShow MoreRelatedAnalysis Of The Poem Battle Royal 857 Words   |  4 Pagesthe elements of a test contribute to a story’s meaning. The images, symbols, and metaphors in Ellison’s â€Å"Battle Royal† give this story a whole new meaning. In 1947, when this short story was published, the black community was at an all-time low. The main aspects of this piece have ambiguous meanings behind them. The grandfather’s death scene, the naked dancer, the battle royal, the narrator’s speech, and the narrator’s dream all have significant symbolism. 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