Differential Gram’s staining Essay Example for Free

Differential Gram’s staining Essay To Gram stain the given bacterial suspension and to differentiate between gram positive and gram negative organism. Visualization of microorganisms in the living state is very difficult, not just because they are minute, but because they are transparent and almost colorless when suspended in an aqueous medium. To study their properties and divide microorganisms into specific groups for diagnostic purposes, biological stains and staining procedures, in conjunction with light microscopy, have become major tools in microbiology. Chemically, a stain may be defined as an organic compound containing a benzene ring plus a chromophore and an auxochrome. Stains are of 2 types: 1. Acidic stains e.g., picric acid 2. Basic stains e.g., methylene blue. Types of staining techniques: 1. Simple staining. (Use of a single stain)This type of staining is used for visualization of morphological shape (cocci, bacilli, and spirilli) and arrangement (chains, clusters, pairs, and tetrads). 2. Differential staining. (Use of 2 contrasting stains)It is divided into two groups: (a) Separation into groups, Gram stain and acid-fast stain. (b) Visualization of structures, Flagella stain, capsule stain, spore stain, nuclear stain. The Gram Stain The Gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between Gram-positive and Gram-negative bacteria. Bacteria that stain purple with the Gram-staining procedure are termed Gram-positive; those that stain pink are said to be Gram-negative. The terms positive and negative have nothing to do with electrical charge, but simply designate 2 distinct morphological groups of bacteria. Grampositive and Gram-negative bacteria stain differently because of fundamental differences in the structure of their cell walls. The bacterial cell wall serves to give the organism its size and shape, as well as to prevent osmotic lysis. The material in the bacterial cell wall that confers rigidity is peptidoglycan. In electron micrographs, the Gram-positive cell wall appears as a broad, dense wall 20–80 nm thick and consists of numerous interconnecting layers of peptidoglycan. Chemically, 60% to 90% of the Gram-positive cell wall is peptidoglycan. Interwoven in the cell wall of Grampositive are teichoic acids. Teichoic acids that extend through and beyond the rest of the cell wall are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Some have a lipid attached (lipoteichoic acid). The outer surface of the peptidoglycan is studded with proteins that differ with the strain and species of the bacterium. The Gram-negative cell wall, on the other hand, contains only 2–3 layers of peptidoglycan and is surrounded by an outer membrane composed of phospholipids, lipopolysaccharide, lipoprotein, and proteins. Only 10%–20% of the Gram-negative cell wall is peptidoglycan. The phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan. The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A: embedded in the membrane, and a polysaccharide portion extending outward from the bacterial surface. The outer membrane also contains a number of proteins that differ with the strain and species of the bacterium. PRINCIPLE The Gram-staining procedure involves 4 basic steps: 1. The bacteria are first stained with the basic dye crystal violet. Both Gram-positive and Gramnegative bacteria become directly stained and appear purple after this step. 2. The bacteria are then treated with Gram’s iodine solution. This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both Gram-positive and Gramnegative bacteria remain purple after this step. 3. Gram’s decolorizer, a mixture of ethyl alcohol and acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex, while Gramnegative are decolorized. 4. Finally, the counterstain safranin (also a basic dye) is applied. Since the Gram-positive bacteria are already stained purple, they are not affected by the counterstain. Gram-negative bacteria, which are now colorless, become directly stained by the safranin. Thus, Gram-positive bacteria appear purple and Gr am-negative bacteria appear pink. With the current theory behind Gram-staining, it is thought that in Gram-positive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violetiodine complex within the cell. In the case of Gram-negative bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The single thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the cell is decolorized. It is important to note that Gram-positivity (the ability to retain the purple crystal violet-iodine complex) is not an all-or-nothing phenomenon, but a matter of degree. There are several factors that could result in a Gram-positive organism staining Gram-negatively: 1. The method and techniques used: Overheating during heat fixation, over-decolorization with alcohol, and even too much washing with water between steps may result in Gram-positive bacteria losing the crystal violet-iodine complex. 2. The age of the culture: Cultures more than 24 hours old may lose their ability to retain the crystal violet-iodine complex. 3. The organism itself: Some Gram-positive bacteria are more able to retain the crystal violetiodine complex than others. Therefore, one must use very precise techniques in Gram staining and interpret the results with discretion. Crystal violet (2gm Crystal violet + 20ml 95% ethanol + 0.8gm Ammonium oxalet + 80ml distilled water) Gram’s iodine (1gm iodine + 2gm potassium iodide + 100ml distilled water) Decolorizer (Mix 95% alcohol acetone in equal proportion) Safranin (0.34gm safranin + 10ml absolute alcohol + 90ml distilled water) Clean grease free glass slide Nichrome wire loop Dropper Filter papers Compound microscope Cedar wood oil Miscellaneous PROCEDURE: On a grease free slide prepare smear of bacterial suspension and allow it to air dry. After drying heat fix smear and allow slide to cool. Cover the smear with crystal violet stain and leave for 1 minute Drain stain and wash the slide carefully under running tap water. Flood the smear with Gram’s iodine solution and wait for 1 minute. Wash the slide under running tap water. 6. Decolorize the smear with alcohol-acetone for 20-30 seconds (continue till purple stain just stops coming off the slide). 7. Gently wash the slide under running tap water and drain completely. 8. Counter stain the smear with safranin for 1 minute. 9. Wash the slide with water and air dry (or dry it carefully by using a blotting paper). 10. A drop of cedar wood oil is put over smear and the slide under oil-immersion lens. 1. 2. 3. 4. 5. Determine the growth curve of Escherichia coli by using colorimeter. The increase in the cell size and cell mass during the development of an organism is termed as growth. It is the unique characteristics of all organisms. The organism must require certain basic parameters for their energy generation and cellular biosynthesis. The growth of the organism is affected by both physical and Nutritional factors. The physical factors include the pH, temperature, Osmotic pressure, Hydrostatic pressure, and Moisture content of the medium in which the organism is growing. The nutritional factors include the amount of Carbon, nitrogen, Sulphur, phosphorous, and other trace elements provided in the growth medium. Bacteria are unicellular (single cell) organisms. When the bacteria reach a certain size, they divide by binary fission, in which the one cell divides into two, two into four and continue the process in a geometric fashion. The bacterium is then known to be in an actively growing phase. To study the bacterial growth population, the viable cells of the bacterium should be inoculated on to the sterile broth and incubated under optimal growth conditions. The bacterium starts utilising the components of the media and it will increase in its size and cellular mass. The dynamics of the bacterial growth can be studied by plotting the cell growth (absorbance) versus the incubation time OR log of cell number versus time. The curve thus obtained is a sigmoid curve and is known as a standard growth curve. The growth curve has four distinct phases 1. Lag phase When a microorganism is introduced into the fresh medium, it takes some time to adjust with the new environment. This phase is termed as Lag phase, in which cellular metabolism is accelerated, cells are increasing in size, but the bacteria are not able to replicate and therefore no increase in cell mass. The length of the lag phase depends directly on the previous growth condition of the organism. When the microorganism growing in a rich medium is inoculated into nutritionally poor medium, the organism will take more time to adapt with the new environment. The organism will start synthesizing the necessary proteins, co-enzymes and vitamins needed for their growth and hence there will be a subsequent increase in the lag phase. Similarly when an organism from a nutritionally poor medium is added to a nutritionally rich medium, the organism can easily adapt to the environment, it can start the cell division without any delay, and therefore will have less lag phase it may be absent. Exponential or Logarithmic (log) phase During this phase, the microorganisms are in a rapidly growing and dividing state. Their metabolic activity increases and the organism begin the DNA replication by binary fission at a constant rate. The growth medium is exploited at the maximal rate, the culture reaches the maximum growth rate and the number of bacteria increases logarithmically (exponentially) and finally the single cell divide into two, which replicate into four, eight, sixteen, thirty two and so on (That is 20, 21, 22, 232n, n is the number of generations) This will result in a balanced growth. The time taken by the bacteria to double in number during a specified time period is known as the generation time. The generation time tends to vary with different organisms. E.coli divides in every 20 minutes; hence its generation time is 20 minutes. 3. Stationary phase As the bacterial population continues to grow, all the nutrients in the growth medium are used up by the microorganism for their rapid multiplication. This results in the accumulation of waste materials, toxic metabolites and inhibitory compounds such as antibiotics in the medium. This shifts the conditions of the medium such as pH and temperature, thereby creating an unfavorable environment for the bacterial growth. The reproduction rate will slow down, the cells undergoing division is equal to the number of cell death, and finally bacterium stops its division completely. The cell number is not increased and thus the growth rate is stabilised. If a cell taken from the stationary phase is introduced into a fresh medium, the cell can easily move on the exponential phase and is able to perform its metabolic activities as usual. 4. Decline or Death phase The depletion of nutrients and the subsequent accumulation of metabolic waste products and other toxic materials in the media will facilitates the bacterium to move on to the Death phase. During this, the bacterium completely loses its ability to reproduce. Individual bacteria begin to die due to the unfavorable conditions and the death is rapid and at uniform rate. The number of dead cells exceeds the number of live cells. Some organisms which can resist this condition can survive in the environment by producing endospores.

Comparing Destiny, Fate, Free Will and Free Choice in Oedipus the King and Antigone :: comparison compare contrast essays

Oedipus and Antigone: Is Fate Determined? Is everything determined? This question has caused fierce debate and has plagued both the science and literary worlds. Fate and Prophecy have both appeared in literature, most notably in Ancient Greek and Roman plays. Two plays that stand out as being based on prophecy are Oedipus Rex and Antigone, both written by Sophocles. Sophocles may have eggagerated certain aspects of fate, but he had many correct observations concerning fate and destiny. I think that everything is determined because free will is just an illusion, time travel depends on it, probability dictates it. In the play Julius Caesar, Cassius tells Brutus, "Men at some times are masters of their fate." But is this true? Can we do anything we chose or is the universe's fate fixed? The answers to these questions we may never know. But we can guess. In Antigone, Creon is faced with a decision. Should he condemn Antigone to death or should he let her get away with a crime? He feels as if he has a choice between the two. But, he doesn't. It was determined that he would put Antigone in a cave and try to get her out after contemplating it carefully. No matter what he did, he could not have defied this. He had the choice between the two, but it was determined that no matter what advice he received from Haemon and Tieresius, he would inevitably choose to put her in the cave. As Oedipus portrayed, you cannot escape fate no matter how you try. The prospect of time travel depends on and proves that everything is determined. If you go back and find out you should have turned left when you turned right and change it, then you may think you have defied fate. But, you are utterly wrong. Most likely, like Oedipus, you will do exactly what was determined, and step right into the prophecy. Doing this will, in fact, create an alternate quantum reality in which it was fixed to happed that way! (If you want to understand this fully, read about Feynman's Sum Over Histories in any of Stephen Hawking's books). The prophet who had spoken to Oedipus about his fate knew what was predestined. If Oedipus had gone forward in time and seen what he had done, he would have tried to avoid that fate.

What Do Women Want?

Trisha Punamiya Professor Kaitlyn McWilliams DISC 1313April 4th, 2108 Title â€Å"What do women want? Seem[s] plain enough: education, respect, to be accepted as the intellectual equals of men, emotional and sexual fulfillment, and marriage.† (Staves 170) Susan Staves uses this powerful description to present the standing and role of women in the 18th century patriarchal society. Jane Austen's Pride and Prejudice boldly and skillfully manages and portrays the dynamics between the men and women in that era. Love, infatuation and compromise are revealed through the relationships of different characters in the novel and the implications that these factors have on their decision to marry. Society placed high expectations on women without providing equal opportunities in terms of education, hence there was always a constant wide disparity in the overall treatment of men and women. Marriage was considered to be an ultimate goal for all of the women, and the convention was to marry well. There was a higher importance placed on economic security as compared to love. Heritance, estate and wealth were considered to be the primary factors, whereas love and connection were condemned to develop over time. While Pride and Prejudice represents all of the stereotypical society norms and compliance of the expectations, it also dares to take a new stand in terms of feminism through the main female protagonist, Elizabeth. Austen's Elizabeth defies social expectations and norms in a patriarchal 18th century, rising above the orthodox women including her counterparts. She, being sensible and wise, strongly justifies her decisions, presenting a new front on feminism. 18th century was considered it be a time where development was seen in the treatment of women, and this novel essentially depicts that improvement. Jane Austen, as Wang and Liu believed, gave all her female characters an impersonal freedom, allowing them to be the focus as well as the reflectors of the narration. Austen concentrated on women's routine life and hence was able to provide the readers with a deeper understanding of the social expectations and norms that the women had to comply with on a regular basis. Making her women the center of the novel, Elizabeth specifically, Austen got rid of the masculine discourse and dominance present otherwise in the society, proving the rather developed and sensible nature of Elizabeth. Austen also allowed Elizabeth to transmit information and by following her journey, viewing things from her perspective enabled the readers to build a more sentimental relationship with her. Female characters were portrayed not only from the outside world, seen by an objective observer, but also from within the character giving an opportunity to reveal their own memories and thoughts. Elizabeth's unaffected charming personality overshadowed the arrogant and wealthy men proving her to be an advocator of independence from societal bounds and from traditional views about marriage that hinder women's self-autonomy. All of Austen's women are distinct individuals and have their own set of ideologies and views on marriage and societal expectations. While, some can be described as sensible and mature, others naà ¯ve and dumb. Elizabeth had a fascinating relationship with all of the characters, through which Austen reveals a substantial amount of insight into the shaping and development of Elizabeth as an individual. Despite being the sensible sisters, who shared similar values and ideologies Jane and Elizabeth were rather different in many ways. Jane waited for the right man all along, marriage for her meant love, passion and meaning. She was willing to forgive Mr Bingley for the mistakes he had committed, accepting his long-anticipated marriage proposal. Jane shared her immense happiness with Elizabeth and wished for her to experience this joy as well. This proves that despite being wise, Jane had a longing desire for Mr Bingley only, and chose to be a silent sufferer during their time apart. She didn't even blame Mr Bingley's sisters or Mr Darcy for the negative role they played in her relationship and even refused accept Elizabeth's views on how a bad influence they were to Mr Bingley. As Reena distinguished, Jane was more aware of the manners of the society and believed that it was necessary for women to marry at a certain age, while Elizabeth rejected men, aspiring to marry someone whom she thought of as an equal in terms of temperament and had a strong romantic connection with. Elizabeth was rather affirmative and outspoken than Jane, who was on the contrary naà ¯ve and kept to herself. Despite being the younger one, Elizabeth had an instinct to protect Jane, this was proved when Elizabeth defended Jane's silence to Mr Darcy and Mr Bingley, when Jane and Mr Bingley's courtship was sidelined. Jane's beauty and charm were somewhat affected by her shy and quiet demeanor; and the outspoken and confident Elizabeth is rather protective of her for this reason. Elizabeth is also a rather fastidious girl who discriminates between people. She was quick in observing the differences between Mr Darcy and Mr Bingley. She quickly understood the nature of Mr Collins whose proposal she rejected without least hesitation. Elizabeth claims that â€Å"There are few whom I really love, and still fewer of whom I think well†. (164) She also declares that she is fed up with the inconsistencies in human beings, showing that she is at times prejudiced herself. Jane, on the other hand, forms a good opinion about others and is undiscriminating in doing so. Elizabeth, the society in extension, believe that Jane is too nice and doesn't have a bad thing to say about anybody. Lizzy says to Jane, â€Å"You never see a fault in anybody. All the world are good and agreeable in your eyes. I never heard you speak ill of a human being in my life.† (53) They believe that because she has such a generous heart and the most affectionate personality, she must be naà ¯ve enough to think of the best in people. The fact that she has captivating manners and an angelic personality, she is hence considered to be the more ideal fit into the 18th century society. Elizabeth has a sarcastic wit which enables her to ridicule some people like Mr Collins. She has the capability to laugh at people's absurdities, as she herself tells Mr Darcy towards the end of the novel. Contrary to that, Jane could never mock or retaliate like Elizabeth would, proving her to be the more ideal and proper woman in terms of the societal expectations. The society hence place the two distinctly; this was also seen with the way Mrs Bennet treated both of them differently, relying on Jane more than on Elizabeth. Through Lydia's elopement, Mrs Bennet demanded Jane's company more than Elizabeth's, and often confronted in Jane about Elizabeth's rejections and what a big mistake she was committing. Because of these differences one can conclude that Elizabeth was the only one who dared to step up against the unfairness of the society, unlike Jane who was equally educated and sensible but didn't just possess it in her character to do so. On the hand, there was large disparity in terms of personality between Lydia and Elizabeth. Being the youngest of the five sisters, she received great amount of attention from their mother. Emotional and immature, Lydia is the daughter who shares her mother's characteristics the most. However, she was the least dear to Mr Bennet, we often see him pass sarcastic remarks on how silly and dumb Kitty and Lydia are. On the other hand, Elizabeth resembles her father the most, being his favorite. She is strong-willed, stubborn, frivolous and foolish. Despite, having the same background and upbringing Elizabeth and Lydia are sheer opposites. Lydia is Jane Austen's way of portraying some of the young girls of that era and satirizing them, while Elizabeth, on the contrary, is Austen's way of depicting the development of women in the male-dominated society. Lydia's interests lie within balls and flirting with the new men in town. Not only does this cause great embarrassment to Elizabeth, but to the Bennet family as a whole. Her main aspiration in life is to attain the attention of men, uniquely different from Elizabeth's character. She elopes with Mr Wickham at the first chance that she gets. Samina and Khattak in their article identify Lydia as selfish for being involved in self-interested schemes to achieve individual rather than collective happiness. Lydia barely knew Mr Wickham or his intentions, she was certain that they were going to get married but was so self-absorbed that she never wondered to consider what kind of a person he is and what is aim was. Unlike, the other female characters, Lydia didn't entirely marry for money or wealth, everyone knew that Mr Wickham wasn't the owner of a large estate. She wanted to fulfill her desire of attraction and physical gratification, not love. Like her mother, Lydia had very little common sense, poor judgement and no consideration or understanding of the consequences that her behavior would have on the reputation of the family, especially her two unmarried older sisters. Society considered her elopement a big crime and the eighteenth century was unapologetic to women as such anyway. Lydia's elopement hence brought disgrace to the entire Bennet family. Mr Collins in his letter to Mr Bennet as a form of condolence wrote that â€Å"The death of your daughter would have been a blessing in comparison to this†. This portrays how ignorant Lydia was, despite knowing that society was extremely unapologetic towards women. It also affected the marriage prospects of the other sisters; the society's notion that the older had to be married before the younger sisters was also disobeyed by Lydia. Lady Catherine used Lydia's mistake to portray her disapproval of Elizabeth's presence in Mr Darcy's life. She embarrassed Elizabeth by saying, â€Å"I am no stranger to the particulars of your youngest sister's infamous elopement. I know it all; that the young man's marrying her was a patched-up business, at the expense of your father and uncles. And is such a girl to be my nephew's sister?† (Austen 358). She fails to be a good sibling to her sister Kitty as well, who adored Lydia and supposedly had a tight knit relationship with her. Kitty was upset that Lydia didn't even bother inviting her. Through the portrayal of Lydia's character and behavior, Austen successfully is able to stereotype women who lacked good sense, decorum, and empathy in that century. In contrast, Elizabeth is a sensible young woman with a sharp wit and respectful nature. She takes pride in her background, despite knowing all their faults. She passes a strong comment to Lady Catherine displaying that humility, â€Å"He is a gentleman; I am a gentleman's daughter; so far we are equal.† (Austen 357) Elizabeth demands respect and hopes to marry someone who would treat her as an equal and would allow her to retain her sense of self. She married for love, however not completely disregarding the need for financial stability in her companion.

Prostitution The World s Oldest Profession - 895 Words

Prostitution, sometimes referred to as â€Å"the world’s oldest profession† (Henslin, pg. 54), is defined by James M. Henslin as â€Å"the renting of one’s body for sexual purposes† (pg. 54). This arrangement, though illegal and socially deviant in most parts of the world, exists universally in many different forms (pg. 54). As a matter of fact, types of prostitutes range greatly in variety from call girls – who are said to be â€Å"the elite of prostitutes† (pg. 58), to streetwalkers – â€Å"who have the lowest status among prostitutes† (pg. 58), to sugar babies -young, physically attractive women who provide â€Å"rich, older men† (Kitchener, par.4) â€Å"†¦with attention (and sex) in exchange for the finer things in life† (par. 4). One of the â€Å"finer things in life†(par. 4) in which sugar daddies are now increasingly providing for their sugar babies is a college education, or, at the very least, the money needed to acquire one. In an article titled â€Å"How Sugar Daddies Are Financing College Education†, Caroline Kitchener of the Atlantic writes of a prostitution website created uniquely for sugar babies and sugar daddies. The website is called Seeking Arrangement. On Seeking Arrangement sugar babies are connected to and can view both the profiles and incomes of various sugar daddies (Kitchener, par. 4). In fact, the website is set up and is promoted basically like any other run-of-the-mill dating/partner seeking site (par. 5), even though money is often times traded for intercourse (par. 5). In designingShow MoreRelatedProstitution : The World s Oldest Profession3535 Words   |  15 Pagesâ€Å"Writing is like Prostitution. First you do it for love, and then for a few close friends, and th en for money.† People for the legalization of prostitution believe prostitution can create positive outcomes for numerous people. However, other people believe legalizing it will hurt more people than it would help. 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