Discuss Moral Values

Societies have ideally tried to uphold higher ethical standards and moral values as the code of conduct for all members. Individuals try to behave according to such standards but in reality what is construed as moral or immoral are generally relative to the values adhered to by the individual (Fisher, 2005). If the person values money and wealth, then being unscrupulous in doing business to gain more profits will not be immoral. On the other hand if the individual values justice and fairness, then his/her behavior would naturally be in accordance with such values ands is expected to be morally upright.In the corporate world there is more room for less moral restraints and unethical behavior. Corporations promote competitiveness and reward those who are able to close the biggest deal or to get the largest customer or the greatest profit which means that individuals will likely pursue these goals without any qualms of whether it is moral or not. For example, the corporate scandals in t he stock exchange was said to have been done to drive the stock price upwards falsely even if it meant cheating the shareholders of their money.It is immoral to cheat or to take advantage of other people but for those in the corporate world, it is the usual way of doing business. Some corporations are so competitive that even employees themselves try to outsmart each other and get on top, even if it meant backstabbing, falsely accusing others or even claiming credit for other people’s work. However, not all companies are run this way, some try to be just and honest, but if the organization does not promote a culture of positive moral values then their employees would really not be required to behave as such.

Applebees Monopolistic Competition

Market structure influences how an organization behaves according to pricing, supply, barriers to entry, efficiency and competition. More specifically, Applebee’s, a nation-wide casual dining restaurant chain, is an organization whose structure is considered to be monopolistic competition. Monopolistic competition is a structure that has many buyers and sellers who sell products that are similar but not identical. Hence, instead of being a price taker, Applebee’s has a downward sloping demand curve. Applebee’s is almost like a tiny monopoly because of the differentiation in the products that they sell. Moreover, Applebee’s has some control over their prices but competition tends to dictate the price range for food and beverages. In addition, it’s relatively easy to enter and exit this market without restrictions. Chili’s, T. G. I. Friday’s, Red Lobster, Outback Steakhouse, Olive Garden and Ruby Tuesday’s are Applebee’s main competitors. In grasping market share and maximizing profits, advertising plays significant role in monopolistic competition. All of these organizations spend millions of dollars in advertising but why? Plain and simple, advertising leads to higher profits. One of the commercials that have Applebee’s has is a 550 calorie meal. In today’s society, consumer are becoming more health conscientious and showing a delicious shrimp and pasta meal for under 550 calories appeals to the consumer’s eye. Now without that commercial, how many people would actually know about this? Definitely, not as many. In continuing on how advertising plays a hugh role in monopolistic, consider yourself driving down the road with a friend and you want to grab a bite to eat. Are you more inclined to stop at Applebee’s, a clean safe and economical eating environment or a place you never even heard of such as Jim Bob’s diner with the sign out in front of the building falling down who has a short order cook that looks like a guy that should be in ZZ Top? Odds are you would be more inclined to stop at the well known Applebee’s because you are familiar with this organization because of advertising. Now that Applebee’s has you in the door, think of the extra revenue that is accrued from ordering drinks? If a restaurant buys a bottle of vodka for $10 and is able to get 22 drinks out of that bottle for $3/drink that would make a $56 approximate profit. If you continue the math, millions of dollars are made because it started out with advertising. References Applebee’s. (2010 March). Retrieved March 25, 2010, from Applebee’s website: http://applebees. com

Comparisons of Inca and Aztecs

The Aztecs had city-states and were people of a stratified society. Each of one of these city-states is ruled by a speaker that is chosen by the pipiltin(the nobility). This speaker would have to sacrifice his own blood regularly to show that he is a true king and has good intentions for his empire. The nobles grew stronger with every conquest. The Aztecs also had a governing council but they weren't all that successful because they had lacked real power. The Aztec system was very successful, because it was aimed at political dominance and not head on control of the people. The calpulli had authority over the government but during the 1st hundred years the emperor took over. The Aztecs were very smart in creating â€Å"flower wars†(to leave a few territories unconquered so that periodic wars could be staged so that both sides could obtain captives for sacrifice) ,because it ensured sacrifices. The Incas believed that their ruler was a living god there to represent the sun god on earth. Everything the Incas did ,religiously and politically, had a religious meaning in it. The Incas had a queen(senior wife of king) and she was believed to be linked to the moon. The Inca believed that integration was very important. By using their language(Quechan) they integrated by teaching it too their conquered peoples. They were smart to adopt the split inheritance from the Mayans. The Inca expansion was closely tied together by ancestor worship. They had developed a state bureaucracy of which almost all of the nobility had played a part in. The Aztecs and Incas are alike because the kings of each of the two peoples were elected by siblings of the royal family. The governing council also had a say so in. Both rulers and kings had a â€Å"right hand man† the Aztecs had a prime minister where as the Incas had a high priest. Both the prime minister and high priest(which both had tremendous power) were usually close relatives of the king. Aztecs' and Incas' highest deity was the sun god. In both empires men and women were mostly equal but since the military virtue was emphasized it gave men more power so it wasn't completely balanced. Women were mostly in the household but they still contributed to the empire. The military in both had great power because they were the backbone of the empires; they supplied the peoples with war captives for human sacrifice. Both of the empires used sacrifice as a political terror. Where the Aztecs demanded mostly tribute as well as some labor the Inca people demanded mostly labor. Both took tribute from their conquered peoples.

Who Own's History Research Paper Example | Topics and Well Written Essays - 1250 words

Who Own's History - Research Paper Example Reference to history is part of humanity. It is quite hard for the society to move without visiting the history of yester years in developing policies today. Historians define history as events or scenes that occurred in the past. Past is not distinct, thereby leading to the question who determines history. The question may look cheap but quite nagging. Politicians make their references from historic events. They take there time to study historic events which had greater influence to human life, develop the event to suite their current conditions and unroll it to the society. This reason has led many people argue that history would reoccur in ones lifetime. Largely, the statement makes great sense in human life. The society owes a lot to history because the governance of the day depends on the governance of the previous day. Many people have taken their time to study what took place before their existence because the life of a man is history. Some documentation about history argues t hat a man would be ignorant if he does not take his time to explore what happened before he was born. The progress that the society boost of could only be realized when history is tabled. For instance, how would a person know his great grand father who died before he was born if history does not exist? Some people fear to study history arguing that historic events are past, yet they fail to recognize that history has influenced their lives today. This paper seeks to answer the question who determines which history is anointed as the true history. The quest for who determines which history is anointed as the true history leads to explorations of events that took place in the past. Eric Foner took his time as an historian to evaluate and determine who own history. He came out with his argument, which indicated that each individual in the society has a role in creating history. Studying history provides an opportunity for owning history. The study of the past helps in shaping the futur e by dictating the actions of today. The validity of this statement lies on the role that the government plays in the society. The government has its powers upon the people, yet it cannot exist without the people. Mr. Foner’s book who own history explores the nature of life in American history, South Africa and Soviet Union. His book provides an insight why there is no socialism in America. He goes a head to point out the importance of history in the community and the world. In the beginning of his book, Foner describes his early life in communist family. He started seeing the social and political gap that existed in America, though people claim that America is a land of liberty. The communist fight against racism influenced his life. Foner’s family had portrayed concern to the blacks whom the American white neglected or viewed as second-class citizens. Foner learnt that a person’s commitment in pursuing social justice would infuse the person’s view into history. Many articles and books that people read provide some element of truth to Foner’s view. Rising in the societal rank would expose a person to be remembered in history. Foner took this opportunity as an insight into studying history and changing the society. He took the road to academic success a means that would bring him to the limelight of history. Foner provides the history of America in his book by pointing out the social issues in the American society. He paints America as a nation that has condoned racialism under the guise of the western styles (Foner 157). His history is against the American, capitalists and democratic zeitgeist. The book portrays Foner as a left thinker. The book points the rise of Ronald Reagan and failure of the government to sustain the economy. He points the

Piano Sonata in D Major Essay Example | Topics and Well Written Essays - 750 words

Piano Sonata in D Major - Essay Example I have chosen to evaluate the different musical elements embedded in this composition because I have always been an admirer of Mozart. Since this composition is considered one of his best works, I find it very interesting and analyzing it is something that I will enjoy. The sonata was divided into 3 distinct movements. Each movement possessed different elements of music such as rhythm and melody, color and texture, and form and harmony. However, it should be noted that these differences only highlights the musical and aesthetic value of the piece. The first movement allegro, meaning fast, is written in a strict sonata form. The sonata form is founded around 1770 and eventually became popular and widely utilized in musical composition during the turn of the 19th century. The beginning movement is divided into 3 sections: the exposition; the development; and the recap. In the exposition, Mozart introduces the first and secondary theme which is also known as the main melody. Next, the development section in which the material presented in the exposition is expanded. In this section, remote chords are introduced. Finally, the movement ends with the recapitulation. The material used in the exposition was re-stated in the home key of the piece following an ABA form. The fir The first movement also portrays a thick orchestral texture. Mozart used double thirds, remarkable scales and tremolos to create beautiful melodic line which is sometimes located in the left hand. The left hand octaves resemble the effect of woodwinds, violins, and the vigorous bowing of the celli and double-basses resulting to create an orchestral effect. Though having a thick orchestral texture, Mozart was still able to control and maintain the serenity and elegance of the music which best characterized his musical style. The Second Movement The following movement, a Rondeau en Polonaise in Andante (meaning slow in tempo), is an elegant structure whose lively thematic melody is embellished every time it returns. In this movement, Mozart used a Polonaise as another material for his composition in which during his time resembles a stately procession. The Third Movement The concluding movement, Tema con variazioni(Theme and Variations) is regarded as one of the finest compositions of Mozart in this genre. The theme was modified in to twelve remarkable variations. Although Mozart altered the theme, he maintained that the thematic idea would still be present and aurally recognizable. The underlying accompaniment of the left hand followed the traces of harmonic progressions stated in the original theme. The emergence of dissonances in each variation which somehow created a new harmony made it quite different. Each of the twelve variations that he wrote based on the original theme can be considered as a best reference for composers and arrangers. The variations exploit a lot of musical and technical devises such as virtuosic runs, crossing of hands, octave doubling and the use of parallel minor key in variation 7, remarkable alberti base and many more. The aria like penultimate variation is the most interesting among the twelve variat

Deities of Sexuality and Fertility Essay Example | Topics and Well Written Essays - 500 words

Deities of Sexuality and Fertility - Essay Example She was meant to titillate and goad men to fight each other over her beauty. A certain element of carnal and sexual lust is associated with her and her sculpture shows her bare-chested, playing and fondling her breasts. Among her stories and practices was the prostitution ritual practiced in her temples. In the Roman temple on Acrocorinth, intercourse with the high priestess, after paying the required offerings, was the accepted method of worshipping Aphrodite (Walter, 1985). Demeter on the other hand nourished earth and called the earth mother or the Goddess of life, fertility and grain. She is portrayed as demure, mother like figure, fully clothed and with a staff in hand. Demeter has given corn and barley to mankind and the skill to till the land and make ti fertile. There is no symbolism of carnality or sexuality but only that of fertility and give rise to life. She has been portrayed with images of harvest, flowers and of life. She was the enemy of Aethon the famine god and put him in the stomach of Erysichthon. Demeter however was not demure but had a number of husbands such as Zeus from whom she obtained Persephone; Poseidon from whom she got Despoina and Arion and many others (Walter, 1985). Persephone was the daughter of Demeter and Zeus and was the consort of Hades.

Compare and contrast the ideologies and aims of VILLA and CARRANZA and Essay

Compare and contrast the ideologies and aims of VILLA and CARRANZA and their impact on the Mexican Revolution ( 1910-17) - Essay Example d his success in the revolution was mainly due to his ability to portray himself as a wise, stern patriarch who was the nations best hope for peace.† (Minster About.com Guide) Villa often opposes dictators during his time and revolted against the current regime twice. He strongly believed in his ways and that Mexicans should be free from oppression of people in power and injustice. Carranza joined the revolution due to high ambitions; he collaborated with Francisco Madero who was the brains of the revolution in 1910. As quoted by Christopher online, â€Å"Carranza was not a true believer in reform and he felt that a firmer hand (preferably his) was needed to rule Mexico.† (Minster About.com Guide) Carranza was more into the politics of the matter. One of his major reasons for joining the revolution was his plans of attaining power for himself. After Madero got assassinated by Huerta, another uprising came; this time led by Carranza himself. He was supported by Villa and Obregon on this one. Villa led his army to winning countless battles with his bravery. With his great charisma he was able to gather an army of 3000 in just a month time. He led them the last time against American Troops, after attacks on Columbus and New Mexico, before he was awarded pardon in 1920. Carranza, on the latter part of the second uprising, was sided by the United States. Villa went to the mountains during this time and was considered a fugitive of the New Mexican government. Little was changed during Carranza’s reign. Jennifer Rosenberg added in her online article that; â€Å"Villa retired from revolutionary life in 1920 but had only a short retirement for he was gunned down in his car on July 20, 1923.†(Rosenberg About.com Guide) After Obregon drove him off of power in Mexico, Carranza was gunned by Rodolfo Herrera in his sleep together with his top supporters in 1920. As Christopher Minster quoted it, â€Å"Herrera was put on trial by Obregon, but it was clear that no one missed

Geology in Hawaii Assignment Example | Topics and Well Written Essays - 1500 words

Geology in Hawaii - Assignment Example Individuals also acknowledge that mountains in Hawaii make travel difficult unless the traveler is in a car at an interstate highway. The geology of a region governs the availability of essential raw materials such as important ore minerals for metallurgy, clay for pottery, building stone (ornamentation and construction), gemstones and gold for decorative arts, as well as sand for glass making. The use of such materials is innately joined to emergent technologies and limited by their quantity, accessibility and quality. The knowledge of geology in Hawaii is defined by the practical extent of a rock or mineral resource, and aid construction of models that can predict its viability. Earthquakes occur all over Hawaii, frequently without noteworthy warning or caution. These geohazards can have extensive effects on human and on the earth’s surface. Localized, small earthquakes may cause no damage that is noticeable and may not even be felt by individuals living in the area affected . In contrast, large earthquakes may cause devastation over a wide area and be felt by individuals a hundred miles. ... When there are heavy rains, some areas of each of the Hawaii Islands are vulnerable to flash floods. Intense rain can turn a quiet, small river into a cascading one that sweeps everything that flows on its way. Lives are also lost in the flash floods every few years; mostly people are swept away in their cars or hikers. Heavy rain may come suddenly and can be difficult to forecast. Flash floods in Hawaii are much common than any other natural disaster, such as hurricanes and tsunamis. It is not long ago that heavy and serious flash floods hit the island. In October 2004, Manoa Valley on Oahu was hit by flash flood and completely soaked the University of Hawaii ground floor at Manoa Hamilton library. In addition to destroying parts of the library, the flood carried away at least 60 homes and caused a damage of almost one million dollars. A six weeks rain period in March 2006 caused flooding in many places in Hawaii. Seven people were killed when a dam on Kauai broke. The rain caused t he Waikiki septic tank system to spill over, resulting in a manure spill that polluted parts of the southern shore of the island for a number of days. On November 23, 1982, two hurricanes (Inki and Iwa) left death and devastation in their wake after passing through Hawaii. Iwa hurricane hit the Islands of Kauai, Niihau, and Oahu. The Iwa hurricane was the first to hit the Hawaii statehood since 1959. The Inki hurricane was the most powerful hurricane to hit Hawaii State in recorded history. Not only are the high winds of a cyclone or hurricane very destructive, but a phenomenon known as storm surge that comes with it causes severe flooding in coastal areas. There are a number of happening caused by volcanic

What People feel when they see Advertisements on Television and Social Essay

What People feel when they see Advertisements on Television and Social Media - Essay Example Advertising industries across the world, however, see social media advertising and television advertising as two different platforms. However, social media and television have a connection when it comes to advertising. Social media influences television into the kind of advertising they have and the time for every advertisement (Campbell et al., 2011). Consumer behavior is the primary purpose of the differences in social media advertising and television advertising. Social media is a community, in general, and the people in social media should feel the presence of any company, organization or product that surrounds them outside social media. It is the same for television as people also surround their lives on TV when they are not working. These people that use television and social media have a particular kind of feeling towards social media and television advertising (Louis, Kerr, & Drennan, 2010). The research proposal is going to focus on the feeling of these people and how they r eact to social media and television advertising. Today’s society has people on social media and television thus advertising for products and services should be on social media. Chi (2011, p.46) describes social media as an association in the middle of brands and shoppers, [while] offering an individual channel and coin for client-focused systems administration and social interactions. The tools that businesses use to communicate with customers have significantly changed, and it is easier for the companies to reach now their customers.

Advanced Sport Management 2 (soccer) Essay Example | Topics and Well Written Essays - 2000 words

Advanced Sport Management 2 (soccer) - Essay Example about the specificity of sport (Weatherill, 2003; 2004), despite their repeated claims that EU institutions are failing to fully recognise the specific nature of sport. Specificity of sport was conceptualised when the UEFAs Executive Committee has approved a document in 2006 presented by the European team sport organisations to the French Presidency of the European Union. Here, the European Commission was asked to finally implement the most effectual means of recognising the specificity of sport within a clear lawful basis. It further states that it should be in accordance with the principles set out in the document, and in juxtaposition with team sport federations. During the meeting set in Vienna, the document was given a go signal by the committee. It holds that the European Council should confirm its steadfast obligation to implementing a concrete definition of the specificity of sport. The European Council, the Executive Committee agreed, declared its unanimous support for the principle of dual-career training for young sportspersons and the concept of minimum numbers of home-grown (locally trained) players, or similar policies, in professiona l teams squads (Arnaut, 2006). Because of sport’s specific nature, it should not be regarded less, thus, it should be set apart from the other fields of business activity. This is why the Nice Declaration was signed recognising the important role of sport in the social, educational and cultural functions. In the declaration, such features must be taken into account when European Community law is applied. Furthermore, it recognises that solidarity between amateur or professional levels is a fundamental aspect of sport. With all of its significance, the independent nature of sports bodies should be supported and protected so that there would be autonomy to organise the sports for which they are responsible. The Declaration further confirms that it is the federation that should continue to be the key form of sporting

Research Paper look for Cognitive development in adolescents

Look for Cognitive development in adolescents - Research Paper Example It is said that teenagers display their true character when they are comfortable and have less fear of punishment and reprisal from their parents. This is most probable when with their friends and confidants. In addition to that, the paper is going to base its interpretation based on the observations that are to be made at a cinema hall that couples up as a fast food and snack outlet. II. Description of Setting The observation took place at a popular fun zone just opposite the main highway junction that leads to various places. This zone experiences a high traffic of youngsters who converge to have fun in the movies, boutique and food court where some grab a quick pizza or French fries as they socialize. The main building faces the front of the zone and has frontal balcony where people can sit and have coffee while watching everything and everyone below. The majority of the population on a normal weekend mid-morning consists of teenagers who range from the age of thirteen to about nineteen. Because of the security of the place and the openness, it is presumed that parents can let the teenagers interact on their own without having much interference. This creates a perfect setting for research because one can observe the happenings without interfering with them. The timing is also important because in the morning, the teenagers are highly energetic but as the noon approaches, there is a lower energy display among the youngsters. The study timing was from about 11 a.m. to about 2 p.m. in the afternoon. III Observation of the Adolescent Group Number of adolescent males and females It is observed that the number of female teenagers were almost double than that of younger boys. It was also observed that more females came in pairs or as larger groups while the younger teenage bouts came with fewer or smaller groups than their female counterparts. Approximate age of adolescents or range of ages Most of the youngsters at the court were approximately in their late teens. It was hard to estimate their ages because some of them might have been in larger bodies than their actual age. It was also observed that younger teenage boys (10-13) were more than their female counterparts. However, they seemed to be in a company of someone older. Pre-pubescent or post-pubescent The prepubescent were not as much in number as their fellow counterparts especially in their teenage years. However, those who were present seemed to be very excited and were characterized by frequent fights cries and intolerable to some extent. It was also noticed that both the male and female kids at this ages were being watched by their guardians who were at the line on the food court or seated next to the o bservation table. It was observed that their interaction with other totally new counterparts was quite fact and it was not long before they began playing and causing trouble for everyone. The post pubescent teenagers on the other hand were more composed in behavior but seemed restless in their own way as Fieldman (2008) also writes. Frequent pacing up and down the fun zone was one off their favorite pastime. The fifteen year old boys were hanging

Collaboration Quiz Essay Example for Free

Collaboration Quiz Essay What are the advantages of having diversity in a collaborative learning environment? Having diversity in a collaborative learning environment opens up more possibilities of learning, you will have more ideas and different views on the situation. The advantages on diversity are the different backgrounds of the group members. Because of this they’ll all have a different way of thinking about the subject at hand. With different attitudes, learning styles, and work ethics it really makes a difference in a collaboration learning environment. One of these advantages is they we all have stories of what we know and or been through. So having information on something you have learned on been through in the past is a valuable input. How might factors such as diversity, attitude, learning, and work styles affect collaboration? The factors of diversity, attitude, learning, and work styles affect collaboration by setting a different mood of the situation. All of these combusts can either be a good thing or a bad thing. Since we all are very different in every way one person may feel as their answer is right and don’t deserve to be changed. The different work styles also affect collaboration for example some people are faster workers than others and are very impatient while others like to take their time and double check their work for error. But this may also be a good thing; since there are so many inputs onto the situation their way or thinking may change and will come to a conclusion on the best answer. How does personal responsibility influence the work and success of a group? I think personal responsibility has a strong influence on the work and success of a group, because a lot of people have families and jobs to take care of and at the end of the day they are very tired and feel to rest. There are a lot of things that can happen unexpectedly that no one can predict that’ll cost you to take time off from classes. This can affect the success of the group because if you do not post in the chat how will anyone know if you have your work of the assignment that needs to be turn in?

Implementation of jit at dell

Implementation of jit at dell IMPLEMENTATION OF JIT AT DELL ASPECTS OF JIT By the 1980s the Japanese had achieved manufacturing greatness by practicing continuous improvement, in that a firm is constantly working to improve in every facet of its business functions. To do this, a firm must always increase quality and look for innovative ways to solve problems. All of these are cornerstones of a modern JIT system. Possibly the single piece of JIT most relevant to a study of supply chain management are the partnerships essential to making JIT truly work. A plant cannot implement a JIT system by itself; it must have the cooperation of its entire supply chain. The supply chain must include suppliers, customers, and every department within the company. The sheer amount of information needed for a JIT system to operate well demands that the information flow of the production control system (PCS) coupled with an up-to-date Order Entry and financial system stretches throughout the supply chain, almost to the point at which an entire supply chain operates as one entity. Lastly, gaining workforce commitment to a JIT lean manufacturing system is important. Without the dedication of the workforce, any endeavor is sure to fail. Workforce commitment can be achieved in several ways. A simple way is to cross train the workforce members outside of their normal business function and help increase an employees problem solving ability. In doing so, a firm is empowering its workforce to think about their function in a new way while looking for ways to improve and giving them an overall view of the entire firm, not just their single job. Only then will the proper inventory needs of the plant be truly capable of coming under control and part of a lean moneymaking enterprise. WEAKNESS OF JIT Just as JIT has many strong points, there are weaknesses as well. â€Å"In just-in-time, everything is very interdependent. Everyone relies on everybody else.† (Greenberg, 2002). Because of this strong interdependence with JIT, a weakness in the supply chain caused by a JIT weakness can be very costly to all linked in the chain. Communication is king in a JIT-rich supply chain. There is a risk involved with JIT when there is a communication breakdown and the company cannot generate the correct inventory needed to keep the just-in-time system running smoothly. CHALLENGE AT DELL Dell is known throughout the world as a leader in supply chain management and just-in-time (JIT) manufacturing. Relying heavily on a vendor managed inventory (VMI) model, Dell has nearly eliminated inventory cost while maintaining a JIT manufacturing strategy. Key to supporting this strategy is the ever changing and increasing number of suppliers needed to manage inventory for Dell. In 2002, Dell recognized that in order to continue scalable growth, significant process innovation would be required. After a thorough partner selection process, Dell issued a request for proposal to evaluate potential solutions to address this challenge. SWOT ANALYSIS Strengths: Direct Model: The Dell Direct model was the â€Å"engine of Dells success† (Rangan and Bell, 5). It gave it an advantage over its competitors as it was very hard to duplicate. The Dell direct model was about â€Å"low cost, direct customer relationships and virtual integration† (Rangan and Bell, 5). It was an efficient distribution system â€Å"characterized by build-to-order manufacturing, and products and services targeted at specific market segments† (Rangan and Bell, 5). Basically, this model allowed customers to call Dell directly and order exactly what they wanted in a PC (i.e., they could customize their PCs). It was only after the order was made that the PC was assembled and shipped to the customer in a relatively short amount of time. This Direct Model led to some other strengths Dell was known to have. Differentiated Products: Because of its Direct model, Dell was able to be described as having a differentiated product. Every PC assembled would be somewhat different from the previous based on customer order. This was an advantage for Dell since it enabled it to differentiate itself from its rivals and gain relatively more supplier power. Customer Service After sale customer service was another one of Dells strengths. A customer with a problem could reach a technical support staff through a â€Å"hotline that was manned 24 hours a day† (â€Å"Matching Dell,† 9). After receiving a call, support personnel would retrieve the file that contained details of the customers computer, and could help the customer solve their problem. In approximately 90% of the cases, the customer and support specialist could resolve the problem over the telephone using the â€Å"diagnostic software installed in the factory† (Matching Dell, 9). Customers were very satisfied with Dell and â€Å"rated Dells sales , products, and services highly relative to the competition† (Matching Dell, 9). JIT: Dells Direct Model allowed Dell to manufacture machines that were â€Å"tailored to customer needs† and based on Just-in-Time inventory (Matching Dell, 7). The company assembled computers based on customer orders and â€Å"held no finished goods inventory of standardized machines† (Matching Dell, 7). Holding no inventory allowed Dell to reduce its costs and have a competitive advantage over its rivals. Economies of scale: Dell is the greatest producer of laptops and therefore has economies of scale in the laptop business. In the United States, Dell had the highest market share in the laptop (and PC business overall) with a 24.8% market share, compared to 11.6% for IBM which was the second highest. Economies of scale allow Dell to have more entry barriers, and hence there was less of a threat of new entrants. Efficient Assembly Line: Dells assembly line was quick and efficient. The production process, from the day the order was made, to the shipping date, took only about a day and a half (Matching Dell, 8). This was an advantage for Dell because customers were satisfied with the amount it took for their product to be shipped to them. Weaknesses: Direct Model: Dells Direct Model could be seen as a weakness from another point of view. Dell assumes that its customers are educated, which is not the case for every customer. Dell does not provide a retail service which allows the customers to see their computers, and purchase them right away (advantage of retail is that it is time efficient from the consumer standpoint). Therefore, the model is limited because it cuts off a substantial part of the consumer market. Low market share in the International Market: Compared to its rivals, Dell had a low market share in the international market. In Western Europe, Dell only had less than a 10% market share, and in other parts of the world, Dell had a market share lower than 5% (Rangan and Bell, 23). This is a weakness because international diversification is very important. Since most of Dells revenues come from the US market, having a low market share in the international market is more risky today because of the effects of economic downturns. Limited laptop customization: Although Dell had an advantage in allowing customization within its PC industry, there was a limit to how much you could customize your laptop. Therefore, Dell was not much differentiated compared to its rivals within the laptop industry. Quality of Display: According to general customer opinion, Dells laptop has a weakness in its quality of display. The physical aspect of Dells laptops in general was not as great as Apple laptops for example, or even its rivals within the PC industry. Opportunities: European Market: Dell has an opportunity to increase its market share in the European market. Dell can increase its market share by providing simpler services to accompany its products. Dell now has approximately 10% of the market share in Europe, and could increase its share to between 20% and 25%. Indispensability of the Internet: The internet is becoming more and more of a necessity. This could be an opportunity for Dell to include wireless options on its laptops and could also consider incorporating Bluetooth. Mobility: Dell could consider focusing on producing more products that allow for mobility. As demand for mobility increases, Dell could take advantage of that to satisfy the customers demand. Growing Economy: Increasing growth in the economy is an opportunity for Dell to increase its market share and at the same time production as well. Since most of Dells revenues come from large businesses, a positive growth in the economy may increase demand for production. Threats: Increasing Demand for Innovation The threat with performance increasing each year is that there is a physical limit to how far you can go. People will soon be satisfied with the level of performance not to demand anything more, and are going to be more susceptible to other things such as prices, or quality of the product. If Dell only focuses on performance, this trend might be a threat in the future. Intensity of Rivalry: As performance increases, differentiation between brand names is going to decrease. Dells rivals are finding ways to increase their market share by replicating some of Dells advantages. For example, IBM recognized the advantages of direct distribution and launched initiatives to expand its own direct sales (Matching Dell, 10). Compaq saw the advantage of reducing inventory, and therefore took initiatives to do so. It â€Å"moved from a production system in which it built business PCs according to its own forecast to one in which it built according to forecasts made by channel members† (Matching Dell, 11). This change in production allowed Compaq to double its inventory turnover (Matching Dell, 11). Since its rivals are starting to â€Å"copy† its strategies, Dells strengths would no longer be advantages if this continues. Inability to innovate: Consumers are constantly looking for improvements in technology. If Dell fails to keep up with consumer demand, it can make great losses, and could lose a major part of its market segment. PDAs replacing laptops: Whether this is a threat or not is still unknown, but there is still a possibility that PDAs will soon replace laptops. Debates are made on whether or not PDAs are complimentary to or substitutes for laptops. If the case was that PDAs substitute laptops, it would be a threat to Dells laptop business. SOLUTION After a thorough evaluation, Dell selected World Wide Technology (WWT) for its ability to leverage key elements of previous supply chain experience, unique end-to-end systems offering and flexibility demonstrated by the proposed approach. Since 2003, WWT has been responsible for the procurement, inventory management and distribution of direct material classified as â€Å"Alpha† products integrated into Dells finished goods. Implementation of the WWT solution fundamentally changed how materials are delivered to Dell. Multiple supplier deliveries have been replaced by a single truck delivery containing all replenishment products arriving in 45 minute intervals. Using radio frequency (RF) devices, WWT Site Coordinators on the factory floor generate demand signals to the collocated distribution center initiating the pick, pack and delivery to the factory just prior to consumption. JIT AT DELL: People In the legacy environment, each supplier was required to provide an on-site resource to maintain inventory levels in the factory. Upon implementation of the WWT solution, WWT became the single partner providing resources on the factory floor, reducing the overall number of resources required to maintain continuity of supply. WWT also built a dedicated team of material planners, vendor managers and process consultants to ensure end-to end management and efficiency of the solution. Process WWT brought several process and technical innovations to the Dell team. First, WWT developed a suite of forecast, inventory and demand planning applications calibrated to fluctuations in Dells manufacturing process. Second, WWT opened distribution centers collocated with each Dell factory reducing lead times to 45 minutes or less. Finally, implementation of a RF based barcode scanning system used for product acceptance, replenishment requests and invoice reconciliation at the factory floor. These innovations while removing cost also provided much greater visibility and reliability relative to continuity of supply. Partners Key to the WWT solution was working with each of the legacy suppliers. Management of 40 suppliers with 40different processes and systems was a key challenge that the team faced. Leveraging technology, the WWT team was able to provide an integrated process for each supplier to receive orders, monitor forecast and reduce personnel cost. Also, by reducing entry barriers for suppliers wanting to work with Dell, WWT has been able to introduce a new level of free market competition further reducing SKU prices for individual components managed by WWT. RESULTS WWT successfully developed the needed systems, opened three initial facilities and put together a support team in just under three months. In addition, the project went live just prior to the holiday season, which is typically the most critical time of year for Dells business. A member of Dells supply chain management team noted, â€Å"Our new program with WWT has created new efficiencies and simplicity in our supply chain. Consolidating supply chain management with a single entity has eliminated a lot of waste.† Dell has been able to acknowledge savings across multiple business areas recognize overall SKU price reductions and experience a scalable process necessary to support Dells overall growth. Since the program began in 2003, WWT has added three additional distribution centers, added more than 50 suppliers, built a team of more than 100 people to support Dell and is expanding into Dells international manufacturing facilities. CONCLUSION: To conclude, in order for Dell to compete efficiently in the laptop industry in the future it needs to take advantage of all of the above opportunities. Consumers are becoming increasingly more demanding and price-sensitive. The next generation laptop must be consistent with the relevant trends affecting the industry today. If Dell succeeds in its attempt to make a product that fits the trends, it will probably still remain in its leading position in the future. REFERENCES: Article: Interview: Dell Pres: Phones, Computers to Stay Separate. The Wall Street Journal, February 18th, 2003.

Review Of Im Nobody! Who Are You?

Review Of Im Nobody! Who Are You? Thesis Statement: Emily Dickinson poem, Im Nobody! Who Are You?, is successful on influencing readers that the best ideas can come from nothing. Introduction: I chose this as one of the famous poets known poem, which is found at Chapter 10.1, entitled: The Speaker (Persona) in a Poem. It can also be found from Thomas H. Johnsons (1960) Book of Complete Poems as its 288th entry: Im Nobody! Who are you? Are you- Nobody-Too? Then theres a pair of us! Dont tell! theyd advertise, you know. - How dreary-to be- Somebody! How public-like a frog- To tell ones name- the livelong June- To an admiring bog! What about the poems form, language, content, or other dimension do you find engaging?: I find engaging its genre of lyrical form, English language using informal diction and bearing a very good rhyme scheme (o sound in lines 1 and 2 with g in 6 and 8) with 2 stanzas. The theme shows how one can find the identity of self and gather the best ideas can come from only doing nothing. It uses the simile and metaphor figure of speeches, because it was able to compare or equate unlike things in similarity (Nobody vs Famous persons) and used the word like in line 6 in the second stanza. Moreover, to dissect the poem, when the word they was mentioned in line 4, wherein the poet was pertaining to famous people or any person situated in high levels the of society (Essay on Emily dickinson i am nobody! who are you? para. 1). With a very short content of only 8 lines, Dickinson was able to quickly reach out to others who would want to have the same privacy she experienced and also gain fruitful o utcomes from this choice. Doing this is actually not seen as usual in famous persons or high level situated bodies. How does the poems use of language compare to that of everyday speech?: I can say that in terms of both modern and classic writing on poetry, it still uses informal diction, though there certainly will be a difference in the use of words if one can write it today. To site a modern view on Dickinsons way of writing in the Im Nobody! Who Are You? poem, Jessica Writes (2007) wrote an online essay saying that, it is evident here that she is referring to a friend that she had relationships kept in private, but were foretold mostly by her corresponding letters. Indeed she is successful in her field. Wherein, she was productive during times she was alone, nursing the gardens inside their yard, writing poetry and reading. Since, she preferred to be named as an anonymous poet to her poems; it shows a personal assessment as A Nobody in the society, being a direct reflection of ideals as a non-conformist to the society. Its psychological nature are greatly affecting to people and objects geared towards seeing themselves in this way in life. With todays social pressures, even the smallest poem can help. Back in the old days, wherein classic literature are given birth to, renaissance and revolutions are greatly affected with writers who seek for a new beginning and freedom thru their writing skills. How do the differences and/or similarities between speech and poetic form affect your experience of the poem?: Domhnall Mitchell (2000), a very good critic of literature, mentioned in his book, that the poem Im Nobody! Who Are You? is a form of confessional poetry. Wherein, I agree with this thought. That is why it uses the simile and metaphor figure of speeches, because it was able to compare or equate unlike things in similarity, which is the Dickinson and akin others being the Nobody type of persons judged against the Famous type of persons (who would not likely to favor practices of a Nobody.) At first, a reader would have taught that Dickinson was talking about herself, but there is a quick turn of mood when the poem becomes about the reader. I find this interesting. This can be seen after the 1 line was said, which is a statement about Dickinsons poetic self, and then there is a following string of questions pertaining to the readers own selves. Lastly, the poems 2 immediate co ncerns are: the readers asking who we are, as well as the how is the relationship existing between the reader and the poet (pp. 157-158). Do these differences and/or similarities influence how you think or feel about the subject matter of the poem?: In terms of familiarity, I can say that this poem is reflective of Dickinsons life and not the persons she sites in her poem. It is more personal in nature. For instance, Arthur Versluis (2001), another very good critic of literature in history, mentioned the opinion of John Cody, in his own book, that her works are pronouncing of her madness as a result. That is why the poem, Im Nobody! Who Are You?, has certain negativity touch on it. It was also quoted, one will inevitably misunderstand and trivialize much of Emily Dickinsons life and poetry if one fails to grasp the full intensity of her suffering and the magnitude of her collapse. For this reason let me state at the onset my thesis that the crisis Emily Dickinson suffered following the marriage of her brother was a psychosis (p. 175). Conclusion: Yes, I agree that the best ideas can come from nothing. You can occupy your time with writing poetries like what Emily Dickinson is known for during her time. Yet, even if one decides a secluded life, society will hunt. Dickinsons writes in such a way that she dictates and forces the readers mind to think the same and view society like her opinion. The advantage I can see here is that it is leading to a self-evaluation and growth in uniqueness as individuals. On the other hand, a disadvantage of her way of writing (as well as thinking) is that it fails to show the right personality for an individual. When, maturity takes place, when one knows his or her purpose in life, then the best ideas can come in, one can make fruitful outcomes and one can reach success. Even the quietest moments or disturbing scenes takes self-confidence and trust, a parallel thought to both being a Nobody and a Famous person.

Heart Of Darkness :: essays research papers

Heart of Darkness ?gHeart of Darkness?h, written by Joseph Conrad, holds thematically a wide range of references to problems of politics, morality and social order. It was written in a period when European exploitation of Africa was at a gruesome height. Conrad uses double oblique narration. A flame narrator reports the story as told by Marlow, assigned to the command of a river steamboat scheduled to transport an exploring expedition. Kurtz is a first-agent at an important trading post of ivory, located in the interior of the Congo. Both Marlow and Kertz found the reality through their work in Africa. Marlow felt great indignation with people in the sepulchral city after his journey to the Congo region because he discovered, through his work, the reality of the universe, such as the great virtue of efficiency, the darkness in society and individuals and the surface reality. When Kurtz found himself on his deathbed and he said ?gThe horror, The horror referring to his life in inner A frica, which caused him disintegration. Marlow emphasized the virtue of ?gefficiency?h throughout the story because he thought of it as the only way to survive in the wilderness. After seeing the dying natives in the forest of the outer station, Marlow described them as ?ginefficient.?h Under ?gthe devotion to efficiency,?h incompetent people were excluded from society. Only efficient people can survive. For example, since Kurtz was the most efficient agent, with regards to producing ivory, his employers respected his achievement and regarded him as an essential person. However, once he fell into disintegration, he was considered no more the than dying natives and thus was treated as if he were dead. He was then buried in the darkness. The symbol of inefficiency was the color green. Marlow illustrated a picture of dying natives, when he said, ?g[They were] black shadows of disease and starvation lying confusedly in the greenish gloom?h(20). Another example of inefficiency is shown i n the description of the body of Marlow?fs predecessor as ?gThe grass growing through his ribs was tall enough to hide his bones?h(13). Marlow realized the real darkness did not existed in Africa but in Europe, and not in Africans but in Europeans who engaged in colonial exploitation, including Kurtz. Due to the natives?f physical features and customs like cannibalism, Marlow defined Africans as the darkness. On the other hand, he considered Europeans as the light because of his illusions of civilization.

Jane’s Path to Prosperity Essay -- Novels Literature Books Religion Pa

Jane’s Path to Prosperity In The beginning of Jane Eyre, Charlotte Bronte introduces Jane as an orphan girl who is residing at Gateshead with Mrs. Reed and her children. Bronte walks us through the episodes of Jane’s life as she moves to Lowood, Thronfield, Moor House, and finally to Ferndean. Throughout these stages, Bronte will show how charity was depicted through the interactions that Jane had. Through these perspectives we will see that the results of how charity was regarded, based on the nineteenth century concepts and views that Bronte discusses, point out the significance of charity in Jane Eyre. Charlotte’s focus on charity in her novel revolves not only around giving money to orphans, but giving love and care to those in need. An example of this would be Graham Gordon’s view that, "Charity is an active concern to help others in their poverty and weakness†¦. This includes not merely alms-giving, or even the giving of emotional support, but sympathetic understanding as well"(159). What is being said is clearly that charity doesn’t only have to do with money, but the thought of acknowledging peoples’ situations in the world with respect. Charlotte’s attitude toward performing good deeds is quite optimistic in her belief that kindness will lead to happiness. In a letter to her friend Ellen Nussey, Charlotte writes, "The right path is that which necessitates the greatest sacrifice of self interest, which implies the greatest good to others; and this path, steadily followed, will lead, I believe, in time to prosperity and to happiness" (qtd . in Winnifrith 51). This piece of information correlates to the Christian belief that this type of action is "the true way to the end" (Graham 10). This clearly states Charlotte’s vi... ...ng Jane to be the ultimate example of what a charitable person is like. We can see clearly how charity was depicted in the nineteenth century and that there were good people who acknowledged the need of others even though they themselves were deprived. The final image that we get of charity in Bronte’s novel through Jane is that no matter what type of people we may encounter in our lives, we can still be capable of reaching prosperity and happiness through our good deeds. Works Cited Bentley, Phyllis. The Brontes and their world. New York: Viking P, 1969. Bronte, Charlotte. Jane Eyre. Boston: Bedford/St. Martin’s, 1996. Graham, Gordon. The Idea of Christian Charity. Notre Dame, In: U of Notre Dame P, 1969. Hinkley, Laura L. Charlotte and Emily. New York: Hasting, 1945. Winnifrith, Tom. The Brontes and Their Background. New York: Barnes, 1973.

Challenges of Leadership Essay

WHAT DO WE MEAN BY THE CHALLENGES OF LEADERSHIP? Being a leader is in itself a challenge. The challenges of leadership are really of three kinds: external, coming from people and situations; internal, stemming from within the leader himself; and those arising from the nature of the leadership role. EXTERNAL CHALLENGES It’s almost impossible to imagine a situation where a leader doesn’t have to cope with external challenges. In an organization, such issues as lack of funding and other resources, opposition from forces in the community, and interpersonal problems within the organization often rear their heads. Social, economic, and political forces in the larger world can affect the organization as well. To some extent, the measure of any leader is how well he can deal with the constant succession of crises and minor annoyances that threaten the mission of his group. If he is able to solve problems, take advantage of opportunities, and resolve conflict with an air of calm and a minimum of fuss, most of the external issues are hardly noticeable to anyone else. If the leader doesn’t handle external challenges well, the organization probably won’t, either. We’ve all seen examples of this, in organizations where everyone, from the director to the custodian, has a const antly worried look, and news is passed in whispers. When people feel that leaders are stressed or unsure, they themselves become stressed or unsure as well, and the emphasis of the group moves from its mission to the current worrisome situation. The work of the group suffers. INTERNAL CHALLENGES While leadership presents to each of us the opportunity to demonstrate the best of what we are, it also exposes our limitations. In many cases, good leaders have to overcome those limitations in order to transmit and follow their vision. Fear, lack of confidence, insecurity, impatience, intolerance (all can act as barriers to leadership. At the same time, acknowledging and overcoming them can turn a mediocre leader into a great one. It’s often very difficult for people, especially those who see themselves as leaders, to admit that they might have personality traits or personal characteristics that interfere with their ability to reach their goals. Part of good leadership is learning to accept the reality of those traits, and working to  change them so they don’t get in the way. Sometimes, what seems to be an advantage may present a challenge as well. A leader who’s extremely decisive may alienate followers by never consulting them, or by consistently ignoring their advice. A leader who’s terrific at developing relationships with others in the organization may be unable to tell someone when she’s not doing her job. Some characteristics can be double-edged swords, positive in some circumstances and negative in others. The real challenge is in knowing the difference, and adapting your behavior accordingly. CHALLENGES ARISING FROM LEADERSHIP ITSELF Real leadership makes great demands on people. As a leader, you are responsible for your group’s vision and mission, for upholding a standard, often for being the group’s representative to the rest of the world and its protector as well. These responsibilities might be shared, but in most organizations, one person takes the largest part of the burden. In addition to its responsibilities, leadership brings such challenges as motivating people – often without seeming to do so – and keeping them from stagnating when they’re doing well. Leaders also have to motivate themselves, and not just to seem, but actually to be, enthusiastic about what they’re doing. They have to be aware of serving their group and its members and all that that entails. In other words, they have to be leaders all the time. WHEN ARE THE CHALLENGES OF LEADERSHIP MOST OBVIOUS? One obvious – and correct – answer to this question is â€Å"all the time,† but in fact some times are more likely than others. Leadership is usually the most difficult when the situation is changing or unstable. When a grass roots group is doing well – gathering allies, getting its message across, attracting funding – no one much notices what the director does; but when something unexpected happens, she’s expected to take care of it, often in a very public way. Some particular times when challenges may arise: * When something new is about to start. When you’re beginning a new intervention, trying something different in a program that’s been running for a while, stepping up to another stage in your initiative, or hiring a new leader, no one is quite sure what’s going to happen. Systems and  relationships can break down, and it’s often a matter of leadership as to whether the new situation is successful or not. * When something is about to end. Often at the end of a school year, a particular project or initiative, a training period – anytime when something is coming to an end and things are, by definition, about to change – times get difficult. That may be because of a big push to get finished, or because it’s tough to tell what’s coming next, or because a close-knit group is splitting up. Whatever the reason, it often takes leadership skills to make sure that the project ends successfully, and everyone moves on to the next phase, wh atever that is. * When times are tough. If there’s not enough funding, or an organization or group is being publicly criticized, for instance, its leader usually has to try to solve the problem in some way: find money, reduce expenses, defuse the attacks. Leaders are tested when times are difficult. * During transitions. There are many ways in which a group can be in transition. It may go – because of a grant or because of other circumstances – from a loosely organized, grass roots collective to a much more formally structured organization. It might grow quickly†¦even too quickly. It might be losing some key people, or changing leaders. One of the most difficult tasks a leader faces is trying to keep a group stable through a period of change. WHAT ARE SOME OF THE SPECIFIC CHALLENGES THAT MANY LEADERS FACE, AND HOW to COPE WITH THEM? EXTERNAL CHALLENGES The world surprises us at every turn, throwing up barriers where the way seems clear, and revealing broad highways where there seemed to be only brick walls. Both kinds of surprises – sometimes the positive more than the negative – present opportunities for exercising leadership, with all the challenges they entail. Some common situations that call for leaders to use their resources include: * Public criticism, especially uninformed criticism, of your group or mission. * Flare-ups of others’ interpersonal issues, either within the group or outside it. * Crises, which could be tied to finances, program, politics, public relations (scandals), legal concerns (lawsuits), even spiritual issues (loss of enthusiasm, low morale). * Disasters. These are different from crises, in that, in a crisis, something important (usually negative, but not always) seems to be  happening, and you’re trying to control the situation. In a disaster, the worst has already happened, and you’re trying to deal with that in some way. * Opposition and/or hostility from powerful forces (business groups, local government, an influential organization, etc.) * A financial or political windfall. Sometimes an unexpected benefit can be harder to handle than a calamity. * Collaboration with another group or organization may call upon a leader to define clearly the boundaries within which he can operate, and to balance the needs of his own group with those of the collaborative initiative as a whole. HOW TO COPE WITH EXTERNAL CHALLENGES Be proactive. Regardless of the situation, it’s important for leaders to do something. Waiting is occasionally the right strategy, but even when it is, it makes a group nervous to see its leader apparently not exercising some control. Be creative. Try to think â€Å"outside the box,† i.e. in unexpected but effective ways. If disaster has struck (you’ve just lost a major source of funding, perhaps ), how can you turn what looks like the end of the world into a new beginning? Can you change the way the organization operates to deal with the loss? Can you use the fact that you’re about to lose services to gain community and political support? Is this an opportunity to diversify your funding? Can you expand your horizons and your reach through collaboration? Don’t just look at the obvious, but consider a situation from all perspectives, and search for unusual ways to make things work. An important piece of information, one that’s often quoted in community work, but which can’t be overstated: the Chinese character for â€Å"crisis† combines the characters for â€Å"danger† and â€Å"opportunity.† Face conflict squarely. This doesn’t mean come out fighting, but rather identify and acknowledge the conflict, and work to resolve it. This is true both for conflict within your group, and conflict between the group and others outside it. Far too many people, leaders included, act as if conflict doesn’t exist, because they find it difficult or frightening to deal with. As a result, it only grows worse, and by the time it erupts, it may be nearly impossible to resolve. If  it’s faced early, nearly any conflict can be resolved in a way that is beneficial for everyone involved. It’s a function of leadership to have the courage to name the conflict and work on it. Always look for common ground. If there’s opposition to what you’re doing, it may only be to one specific part of it, or may be based on misunderstanding. There are few groups or individuals who don’t have some common interests. If you can find those, you may have a basis for solving problems and making it possible for people to work together. Retain your objectivity. If you’re mediating a conflict within the organization, don’t take sides, even if you think you know one side is right. That will come out if you mediate objectively and well.If you’re faced with detractors or opposition, don’t automatically assume they’re villains. What are their concerns, and why do they disagree with what you’re doing? Don’t get sucked into a fight unless there’s really no alternative. Even rabid opposition can often be overcome through a combination of respect, political pressure, and creative problem solving. When you do feel you have to fight, pick your battles carefully. Make sure you have the resources – money, political and other allies, volunteer help, whatever you need – to sustain conflict. Battles can advance your cause, or they can kill your initiative once and for all. Don’t get into a fight you have no chance to win. Look for opportunities to collaborate. This is important both within and outside your group or organization. Within the group, involve as many people as possible in decisions, and make sure they have control over what they do. The more they own their jobs and the organization, the more enthusiastic they’ll be, the more effective the organization will be, and the more effective you’ll be as a leader. Outside the organization, try to forge ties with other organizations and groups. Let them know what you’re doing, get and give support, and work with them to the extent you can. Make common cause with other groups that have similar interests. In numbers, there is strength, and you’ll be stronger as an alliance of groups than any one of you could be individually. INTERNAL CHALLENGES Leaders are human. That’s hardly news, but it means that they come with all the same problems and failings as everyone else. One of the greatest challenges of leadership is facing your own personal issues, and making sure they don’t prevent you from exercising leadership. Acknowledging the attitudes and tendencies that get in your way, and working to overcome them, is absolutely necessary if you’re to become an effective leader. Among the most common personal traits that good leaders have to overcome or keep in check are: * Insecurity. Many people feel, at least some of the time, that they’re not up to the tasks they face. They may even believe that they’re fooling people with their air of competence, when they know they’re really not very capable at all. Insecurity of that sort keeps them from being proactive, from following their vision, from feeling like leaders. It can be crippling to both a leader and her group or organization. * Defensiveness. Also born of insecurity, defensiveness shows up most often as an inability to take criticism (other people might catch on to the fact that you’re as incompetent as you know you are), and continuing hostility to anyone, even an ally, who voices it. Defensiveness often also includes a stubborn resistance to change ideas, plans, or assumptions, even if they’ve been shown to be ineffective. * Lack of decisiveness. Sometimes it’s hard to make a decision. You never know till later – and sometimes not even then – whether you made the right decision. Maybe if you had a few more facts†¦ The reality is that leaders are called on to make decisions all the time, often with very little time to consider them. It is important to have as much information as possible, but at some point, you just have to make the decision and live with it. Some decisions are reversible, and some are not, but in either case, it’s important to learn to make a decision when necessary and understand that living with the consequences is part of being a leader * Inability to be direct when there’s a problem. Many people want so badly to be liked, or are so afraid of hurting others, that they find it difficult to say anything negative. They may be reluctant to tell someone he’s not doing his job adequately, for instance, or to address an interpersonal problem. Unfortunately, by letting these things go, they only make them worse, which makes them still harder to address. It’s essential to learn when firmness is necessary, and to learn how to exercise it. *  Inability to be objective. Neither looking at situations through rose-colored glasses nor being always on the edge of hysteria is conducive to effective leadership. Just as objectivity is important in dealing with external issues, it’s important to monitor your own objectivity in general. There’s a difference between being an optimistic individual and being unable to see disaster looming because it’s too painful to contemplate. By the same token, seeing the possible negatives in an apparently positive situation is not the same as being paralyzed by the assumption that calamity lurks around every corner. The inability to accurately identify the positive and negative in any situation and react appropriately can create serious problems. * Impatience – with others and with situations. It may seem, given the importance of decisiveness and firmness, that patience is not a virtue a leader needs. In fact, it is perhaps the most important trait to develop. Situations do not resolve themselves instantly, and anyone who’s ever been involved in an organization knows that Rule #1 is that everything takes longer than you think it will. People in unfamiliar situations need a while to orient themselves. Leaders who are impatient may make rash decisions, may alienate staff members or volunteers or allies, and can often make situations worse rather than better. It’s hard to be patient, but it’s worth the effort. In addition to character traits that can get in a leader’s way, there are the effects of health and personal crises. COPING WITH INTERNAL CHALLENGES Listen. Listen to people’s responses to your ideas, plans, and opinions. Listen more than you talk. Listen to a broad range of people, not just to those who agree with you. Probe to find out why they think or feel the way they do. Assume that everyone has something important to say. If you hear the same things from a number of different and diverse sources, you should at least consider the possibility that they’re accurate. If they’re about things you do that you can change, you might give it a try. Ask for 360-degree feedback†¦and use it. This is feedback (people’s views of you) from everyone around you – staff, volunteers, Board, participants, people from other organizations or groups  yours works with – anyone you work with in any way. As with listening, if you hear the same thing from a lot of different sources, it’s probably true. Act on it. All the feedback in the world won’t do you any good unless you do something with it. Look at what’s going on around you. Are you the center of controversy and chaos? Or do calm and good feeling seem to reside wherever you do? The chances are that the answer lies somewhere in between these extremes, but it probably should be closer to the calm and good feeling side. Even if you’re involved in a battle with the forces of evil, you can foster calm in yourself and those you work with. At the same time, your group could be on top of the world, and you and your colleagues could still be climbing the walls if that’s the kind of atmosphere you create. Reach out for help in facing internal challenges. Most of us find it difficult to change entirely on our own. A psychotherapist, a good friend, a perceptive colleague, or a trusted clergyman might be able to help you gain perspective on issues that you find hard to face. Many people find meditation or some form of self-discovery helpful in understanding themselves and in getting through change. Don’t feel you have to do it all on your own. CHALLENGES STEMMING FROM THE NATURE OF THE LEADERSHIP ROLE A leadership position brings with it unique demands. Leaders can be looked on as authority figures, as saviors, as fixers of things that are broken, as spiritual guides, as mentors, as models, as inspirers, as teachers†¦in short, they may be seen however others choose to see them. This in itself carries a set of challenges, in addition to those posed by what all leaders indeed have to do in order to keep things going. Some of the issues that leaders have to cope with specifically because they’re leaders are: * Keeping an eye on, and communicating, the vision. As the guardian of a group’s vision, it’s up to the leader to remind everyone of what that vision is, to keep it in mind in everything the group or organization does, to protect it from funders or others who would try to change it†¦and to make sure It does change, if necessary, with changes in circumstances, the needs of the target population, or the available information. That means not being  d istracted from the bigger picture by day-to-day issues (even as those issues are addressed and resolved). It also means not substituting another, lesser goal (getting enough funding to start a specific program, for instance) that may be contrary to the true vision of the organization. * Keeping the everyday under control while you continue to pursue the vision. You can’t maintain the vision without making sure that there’s paper in the printer, that you understand the legal implications of an action you plan to take, that people know what they’re supposed to be doing on a given day, that there’s enough cash in the bank to meet payroll, and that there’s someone there to answer the phone, to pay the bills, and to look for funding. These aren’t necessarily all things a leader has to do herself (although there are certainly organizations where that’s what happens), but she’s responsible for making sure they get done, and that things run smoothly. No matter how transformative she is, no leader can accomplish much if the infrastructure do esn’t work. * Setting an example. If you want others in the group to show mutual respect, to work hard, to embrace the vision and mission of the organization, to include everyone in their thinking and decisions, you have to start by doing those things yourself, and behaving in the ways you want others to behave. A leader who yells at people, consults no one, and assumes his word is law will intentionally or unintentionally train everyone else in the group to be the same way. A leader who acts collaboratively and inclusively will create an organization that functions similarly. * Maintaining effectiveness over time. One of the hardest lessons of leadership is that you’re never done. No matter how well things go, no matter how successful your group or organization or initiative is – unless it’s aimed at accomplishing a very specific, time-limited goal – you have to keep at it forever. Even if you get a bill passed or manage to get money for your cause included in the state budget, you have to work to maintain your gains. If you’re running a community intervention, you have to recruit participants, refine your methods, do community outreach, raise funds†¦indefinitely. Maintaining effectiveness is a matter both of monitoring what you do and working to improve it, and of keeping up enthusiasm for the work within the group. It’s part of the leader’s role to maintain his own enthusiasm and drive, and to communicate and transfer them to others. * Avoiding burnout. This is a challenge not  only for leaders, because a burned out leader can affect the workings of a whole organization. Leader burnout is a product of being overwhelmed by the workload, the frustrations, the stress, and the time demands of the position, multiplied by the number of years spent in it. It can reach a point where the leader no longer cares about the vision, the work of the group, or anything but when he can go home. By that point, the rest of the group is likely to be struggling, feeling rudderless and uncertain. It’s crucial that leaders learn to recognize the signs of burnout and – depending on where they are in their lives and a number of other factors – either find ways to renew their commitment or leave. Perhaps even more threatening than burnout is â€Å"burn-down† – the loss of passion and intensity that can come with familiarity and long service. You may still care about what you’re doing, but the en thusiasm just isn’t there anymore. In many ways, this condition may be even harder to deal with than burnout. At least if you’re burned out, it’s obvious: if you’re burned down, especially if it’s happened over a long period, neither you nor others may have realized it. * Finding support. Cliches often become cliches because they’re true. It is lonely at the top, largely because a good leader tries to make things go smoothly enough that others aren’t aware of the amount of work she’s doing. The leader may have no one to share her concerns with, and may have to find her own satisfaction, because others don’t recognize the amount and nature of her contribution. The buck may stop with her, but where then does she unburden herself? As mentioned earlier, leaders are human. They need support and comfort as much as anyone else, and it’s important that they find it. COPING WITH CHALLENGES STEMMING FROM THE NATURE OF THE LEADERSHIP ROLE So how can you continue to be a leader and also continue to be a functioning human being? There are things you can do to retain both your sanity and your competency. Create mechanisms to revisit your vision. Hold occasional meetings and at-least-yearly retreats to discuss vision and renew commitment. These will serve both to review the vision to see if it still resonates (and to rework it if necessary), and to renew your and others’ purpose and pursuit of it. They’ll help to remind you of why you’re doing this in the first place, give you an opportunity to work on group solidarity, and – ideally – leave you feeling refreshed and ready to carry  on. Share the burden. Surround yourself with good people who share your vision. If you can find others who are competent and committed to whom you can delegate some of the tasks of leadership, it will both remove pressure from you, and make your group stronger. One of the greatest mistakes a leader can make is to be threatened by others’ abilities. In fact, sharing responsibility with capable people makes all of you more effective, and strengthens your leadership. Having competent people to depend on also means that you can develop systems and know they’ll work. Organizational maintenance becomes much easier, and you have more time to devote to the actual pursuit of your vision. Find an individual or group with whom you can discuss the realities of leadership. In many communities, some heads of organizations meet on a regular basis to talk about the difficulties and rewards of their situations with others who truly understand. Some such arrangement can be a valuable hedge against burnout, and can also help you gain insight into how you function as a leader. It can introduce you to alternative ways of doing things, as well as giving you a chance to vent, and to realize you’re not alone. Make sure you have personal time. The founder and director of a prominent think tank once went seven years without a day off – including Sundays. That’s 2,557 straight days of work. (That includes two leap year days, for those of you doing the math.) Even if that doesn’t cause burnout, it’s not good for your creativity or your understanding of the world. Everything becomes work or related to work: the world holds no other reality, and leadership becomes all you do. In order to maintain perspective and to keep yourself fresh, you need to take time away from being a leader, and away from your organization or initiative. It’s important to have an activity that gets you away from your daily concerns, and to take days off from time to time. Some people meditate every day, others play music regularly, others participate in sports or fitness activities. Your getaway doesn’t have to be an everyday thing, but it should be something you love and look forward to, and it should be frequent and regular. It may be as simple as taking a walk with your kids for an hour every evening – whatever it is that relaxes your mind and feeds your soul.  Rather than detracting from your effectiveness, your time off will increase it. Depending upon how you approach it, leadership can be a hard and lonely road, or an exciting and collaborative trip to a new place. The more, and more useful, strategies you can find to cope with its challenges, the better leader you’ll be.

Inoculation of an Egg

1. EGG INOCULATION The fertile hen’s egg can be used to cultivate and propagate various types of viruses. Because of the ability to alter their tropism and to adapt to a new host species, many viruses become capable of growing in chick embryo tissues wherein they frequently attain a much higher concentration than in the tissues of the natural host. STRUCTURE OF AN EGG The extra-embryonic membranes of the chick embryo arise from three germinal layers: the endoderm, mesoderm and ectoderm (Fig. 1).The dorsal somatopleure consists of ectoderm on one side and mesoderm on the other side while the splanchnopleure consists of mesoderm and endoderm. By a process of folding, the somatopleure gives rise to the chorion and amnion while the allantois and yolk sac membranes develop from the splanchnopleure. The amnion arises from the head and caudal regions of the embryo, the membrane being reflected back to form the chorion. the amniotic membranes grow rapidly and fuse to form the amniotic sac by the 5th day. The allantois grows out as a bud from the hind gut of the embryo and enlarges rapidly.By the 10th day the allantois becomes attached to the outer layer of the amniotic sac and the inner layer of the chorion to form the chorioallantoic sac (CAS) which separates the chorion from the amnion. The fused chorionic and allantoic membranes are referred to as the chorioallantoic membrane (CAM). Because the CAS represents a diverticulum of the gut, it serves as the excretory receptacle for the embryo. It contains from 5 to 10 ml of fluid with dissolved solids, the solution being clear in early stages but becoming turbid after the 12th day due to the presence of urates.The CAM is the respiratory organ of the embryo and thus is richly supplied with blood vessels. The embryo is surrounded by the amniotic sac and lies bathed in about 1 ml of amniotic fluid. The amniotic fluid, which contains much of the albumin in the egg, serves as a source of protein which is ingested durin g swallowing movements the embryo is seen to make from the 9th day onward. The air-sac is present in the blunt end of the egg. Underlining the shell is the fibrous egg shell membrane. In the beginning stages of development, the chick embryo can be recognized with difficulty as a small dark area attached to the yolk sac.After 4-5 days the embryo can be readily detected by candling. After the 10th day, the embryo development, rapidly increase in size and feathers appear. The respiratory tract develops between the 12th and 15th days. If the egg remains uninoculated and is maintained in a humid 38oC environment, it will hatch on the 21st day of life. Inoculation Procedures The methods described below for the inoculation of the chick embryo do not comprise a complete list but represent those that are practiced most commonly. Likewise, while there are a number of techniques for inoculation by each of the routes listed, only the one most widely used is described.A. Yolk Sac Chlamydia and r ickettsia grow readily in the yolk sac (YS) membranes. Although some of the smaller viruses are inoculated by the YS route, they invade and replicate in the tissues of the embryo itself rather than in the YS tissues. a. Candling and drilling. Fertile eggs that have been incubated for 5 to 7 days are suitable since the YS is relatively large at this time. The eggs are candled and the boundary of the air sac penciled in. The shell over the air space, which is referred to as the shell cap, is disinfected by an application of iodine to one small area.When the iodine is dried, a hole is made through the shell over the center of the natural air space by means of a drill or egg punch. b. Inoculation and incubation. By means of a syringe fitted with a one and one-half to two inch 23 gauge needle, the inoculum is deposited in the YS by passing the needle through the hole in the shell cap and directing it downward to its full length parallel to the long axis of the egg. From 0. 2cc to 0. 5cc is usually inoculated. the hole in the shell is then sealed with tape and the eggs are incubated at 37oC. c. Harvesting Procedure.The egg is placed in a container which maintains it in the upright position during the harvesting procedure. The shell is cracked with sterile forceps and the cap lifted off. The exposed membranes are torn away. If the YS membranes are to be harvested. The contents of the egg are quickly emptied into a sterile petri dish. The YS is usually ruptured in the process. The YS membranes, which are easily recognized by their deep yellow color, are detached from the embryo and separated from the chorioallantois with sterile forceps and quickly transferred to a sterile petri dish.When the embryo is to be harvested, it is withdrawn by hooking the curved end of a dental probe around the neck. It is then separated from the adherent membranes with sterile scissors and transferred to a sterile petri dish. B. Chorioallantoic Sac (CAS) The influenza and the newcastle dis ease viruses and most other viral agents which cause respiratory infections grow readily in the endodermal cells of the allantoic sac wall and are liberated into the allantoic fluid. The encephalomyelitis viruses and the mumps virus also multiply readily when inoculated by this route. . Candling and drillings. Embryonating eggs which have received a preliminary incubation from 9 to 11 days are candled and the boundary of the air space penciled in. The eggs are held in the upright position with the air sac uppermost. A point is selected a few millimeters above the floor of the air space on the side of the egg where the chorioallantois is well-developed but free of large vessels. Iodine is applied to the area around the site. A hole is then drilled or punched through the shell. b. Inoculation and incubation.A one-half inch 26 gauge needle, fitted to a small syringe containing the inoculum, is inserted into the allantoic cavity by passing it through the hole in the shell parallel to th e long axis of the egg or at an angle directed towards the apical extremity. From 0. 1cc to 0. 2cc of inoculum is injected into each egg. The hole in the shell is then sealed with tape and the eggs are incubated. c. Harvesting of allantoic fluid (AF). In order to avoid hemorrhage into the AF while harvesting, the eggs are chilled in the refrigerator from 4 to 6 hours prior to the harvesting procedure.While harvesting, eggs are held in an upright position and the shell over the air sac is removed with sterile forceps. The floor of the air space is exposed. With a pair of small sterile curved forceps these membranes are torn away. In order to facilitate the harvesting of the AF the embryo is displaced to one side by placing the forceps against the embryo with the tips toward the shell wall. The AF can then be readily aspirated with a 5 ml or 10 ml sterile pipette. C. The Chorioallantoic Membrane (CAM) Nine to 12 days old embryonating eggs are candled and an area over the most vascular portion on the side of the egg marked with a pencil.The shell is disinfected with iodine over this point and also the air sac end. A hole is carefully punched over both these locations. The hole on the side of the egg must penetrate both the shell and the inner shell membrane. A small amount of fluid may exude from the hole if the inner shell membrane is penetrated. While candling the egg with its long axis in the horizontal position, a piece of rubber tubing is placed firmly over the hole in the end of the egg. Suction is applied until the air sac collapses in the end but reappears on the side of the egg.When this false air sac is confirmed by candling, the CAM is ready for inoculation. The CAM and inner shell membrane are usually tightly adherent by 9 days of incubation, and the inner shell membrane may consequently by dropped as well as the CAM. This is unacceptable since the inoculum will fall on the inner shell membrane and not the CAM. To avoid this, a drop of sterile PBS is placed over the newly punched hole in side of egg to soften the membranes. An alternate method is to drop the CAM at 7-8 days of incubation, then wait until 11-12 days before inoculation. a. Inoculation and incubation.By carefully passing the needle through the inner shell membrane from 0. 1cc to 0. 2cc of inoculum is dropped on the chorioallantois with a 1cc syringe fitted with a 22 or 23 gauge one-half inch needle. In very critical studies the egg should be candled during this procedure to insure that the inoculum is deposited on, rather than through, the membrane. After inoculation the egg is gently rocked in order to spread the inoculum uniformly over the surface of the CAM. The opening in the shell is covered with a small square of scotch tape and the inoculated eggs are incubated in a horizontal position with the hole uppermost. . Harvesting of the membrane tissues. The egg is placed in the horizontal position with the hole uppermost. Iodine is applied to the area around the w indow with a cotton swab and the tape then peeled off. The surrounding shell is broken away with sterile forceps and the chorioallantois exposed. The membrane is grasped with forceps, detached with scissors and quickly transferred to a sterile Petri dish. D. Amniotic Sac This method is used principally for the isolation of the influenza virus from throat washings. The embryo during the course of its development wallows the amniotic fluid, thereby bringing the inoculated virus which it contains into contact with the tissues of the respiratory and intestinal tracts where multiplication presumably occurs. After incubation amniotic fluid is then â€Å"subpassaged† by the CAS route (Fig. 2). The amniotic route of inoculation is used also for the isolation of the encephalomyelitis virus. a. Candling and drilling. Embryos from 13 to 15 days of age are used. The position of the embryo is determined by candling and a point on the shell over the air space on the side of the egg on whic h the embryo is situated is marked.The site is prepared in the usual manner and a hole is drilled or punched as for yolk sac inoculation. b. Inoculation and incubation. A 1cc syringe fitted with 1 3/4 inch 24 gauge needle is used for the inoculation. The egg is placed horizontally on the candler, the needle is introduced and gently stabbed in the direction of the embryo. Penetration of the amniotic sac is indicated by a sudden movement of the embryo. The needle is then withdrawn slightly and from 0. 1cc to 0. 2cc of the inoculum injected. the hole in the shell is sealed with tape and the eggs are incubated in the vertical position. . Collection of amniotic fluid. The shell is removed as for the allantoic and yolk sac routes of inoculation. A few drops of saline are placed on the floor of the air space to render the membrane transparent. Using the eyes of the embryo as a reference point, the amniotic fluid is aspirated by means of a Syringe fitted with a short 23 gauge needle. E. Mis cellaneous Routes ofInoculation a. Intravenous. This method is not used commonly, although it is the method of choice for the isolation of bluetongue virus. A large vein is located and marked in 12-14 day embryos.A rectangular piece of shell directly over the vein is removed and a droplet of sterile mineral oil is placed on the inner shell membrane so as to render it transparent. A 27-30 gauge five-eight inch needle fitted to a small syringe is introduced through the membrane into the vein in the direction of blood flow. From 0. 1 to 0. 5cc of inoculum is then injected. Incubation and harvesting of the embryo is carried out as already described. b. Intracerebral. This route may be used in the studies of pathologic alterations of the brain following infection. Eight to 14 day embryos are usually used.The viruses of herpes simplex and rabies may be cultivated by this method. Egg Inoculations. Materials needed: Embryonated eggs, 11-12 days A paramyxovirus, PI3 or Sendia virus Vaccinia virus Crystal violet Appropriate syringes and needles Egg candlers, egg punches Iodine disinfectant and swabs, cellophane tape Instruments, petri dishes Procedure: 1. Inoculation of dye into CAS a. Candle 11-12 day embryonated egg, mark boundaries of air sac with pencil. Just above air sac, choose a point devoid of vessels and mark with a pencil. bDisinfect egg shell at this point with iodine.Let dry before next procedure. c. Drill a small hole with an egg punch at the appropriate marked point (be careful-don’t break the shell). d. Inject 0. 1 – 0. 2ml dye into the CAS as described and illustrated. e. Place a small piece of cellophane tape over the hole. The egg would be ready to incubate if the inoculum had been virus. f. Candle the dye-inoculated egg to establish that the inoculum is in the correct place. Watch the inoculum spread through-out the confines of the CAS. g. Break the egg and pour the contents into a petri dish. Observe where the dye is.Identify the CAM, YS, amnion and embryo. If inoculated properly only the CAS should contain dye. 2. CAS inoculation of virus a. Follow procedures for CAS inoculation of a dye, except the inoculation should be 0. 1 ml of a live paramyxovirus. Place tape over the inoculation hole and incubate. b. Candle egg daily to determine embryo viability. If the embryo dies within 2 days of inoculation, it usually indicates bacterial contamina-tion or trauma. c. If the embryo dies after 2 days, refrigerate as soon as death is noted until the next laboratory period. 3. CAM inoculation of vaccine virusWarning;If you have not had a successful smallpox vaccination, have eczema or evidence of immune deficiency, contact the instructor before handling this virus. Be careful! Do not get this virus in your eyes! Vaccinia virus is the live virus vaccine for smallpox. While less pathogenic than smallpox or variola virus it can still cause serious or uncomfortable lesions if mishandled. a. Two 11-12 days embryonated eggs will be supplied to each group of students. Drop the membrane on both of the eggs according to the instructions and illustrations. b. Inoculate 0. ml vaccinia virus onto the dropped CAM. be sure to go through the inner shell membrane but not through the CAM. Rock the egg to distribute the inoculum over the entire floor of the false air sac. Cover the hole with tape and incubate in a horizontal position with the hole uppermost. 4. Harvesting of embryonated eggs (next laboratory). a. Follow instructions for removal of CAS fluid. Try to keep blood vessels from rupturing. Remove CAS fluid aseptically in a sterile pipette. Expel fluid into a sterile vial. This fluid will be used for the hemaggulatination exercise later.It can be frozen if necessary. Use the last drop of CAS fluid to inoculate bacteriological media to check for contamination. b. Harvest CAM as per instructions. Place the membrane in a petri dish and lightly pour PBS over the membrane until it flattens out and the pocks are cl early visible. c. Important. All fluids, instruments, and other things that have come into contact with virus-infected tissues must be properly sterilized. Follow carefully the instructor’s remarks for proper disposal of all materials. Be sure to disinfect your workspace with disinfectant when cleaning up. 1. INFECTIVITY ASSAYS The concentration of a suspension of virus is usually determined by measuring its infectivity. There are two types of infectivity titrations: the quantal assay, which depends upon an all-or-none does response, and the quantitative assay, which utilizes a plaque, pock or lesion count in which the effect of a single infectious virus particle is seen as a visible localized change in a background of normal cells. A. Quantitative Assay2 This method determines the actual number of infectious units (virus particles) in a given suspension.This type of enumerative response is assessed from focal lesions such as plaques in cell cultures, pocks on the CAM of chic k embryos or local necrotic lesions on a plant leaf. The number of infectious units per unit volume can be calculated, and this is referred to as the titer. With plaque assays, the titer of the original virus suspension is stated in terms of the number of plaque forming units (PFU) per ml. Ex. Fifty plaques on a 10-5 dilution of original suspension were counted. A 0. 1ml inoculum was used. #PFU/ml. of original volume No. of plaques = ——————————— dilution) x (Vol. of inoculum) =5. 0 x 107 PFU/ml=50 x 106 = or 105 x 0. 1 B. Quantal Assay This assay estimates the concentration of infectious particles by allowing them to replicate in a suitable host so that one infectious unit can be detected by the amplification effect of the infection. The actual number of infectious particles introduced into the test unit is unknown and may vary even between duplicates of the same dilution. To determine quantal infectivity t iters, mutiple replicate tests are used for each dilution of original suspension until the infectivity is diluted out.The result gives the dose necessary to produce a defined response. This response is usually based on a 50% end point, which is the dilution at which 50% of the test animals, eggs, or cell cultures react to the virus. Computation of the 50% end point is based on the presence or absence of a predetermined criterion, i. e. death (Median Lethal Dose or LD50), infectivity (Median Tissue Culture Infective Dose or TCID50, Median Egg Infective Dose or EID50), etc. The criterion must be either present of absent: either the animal is dead or alive, or the cell culture is infected or not infected.There are no plus/minus or graded reactions. This method does not measure the exact number of virus particle but only whether or not virus is present at a particular dilution. There are two formulas that can be used to determine 50% endpoints; the Reed-Muench and the Spearman-Karber me thods. Both are demonstrated here using the same data. 1. Reed-Muench Method Accumulated Values |Virus Dilution |Morality Ratio | | | | | | | |(a) |(b) |Died |Survived Died |Survived |Ratio |Percent | | | |(c) |(d) |(e) |(f) |(g) |(h) | |10-1 |6/6 |6 |0 |17 |0 |17/17 |100 | |10-2 |6/6 |6 |0 |11 |0 |11/11 |100 | |10-3 |4/6 |4 |2 |5 |2 |5/7 |71 | |10-4 |1/6 |1 |5 |1 |7 |1/8 |13 | |10-5 |0/6 |0 |6 |0 |13 |1/13 |1 | | | | | | | | | | At the 10-3 dilution, 5/7 or 71% of the accumulated test animals died, and at the 10-4 dilution, 1/8 or 13% died (columns g and h). The 50% endpoint, therefore, lies somewhere between the 10-3 and 10-4 dilutions. The final calculation requires interpolation between these two values. The formula for doing this is: (% mortality at dilution next above 50%) – (50%) ————————————————————— = proportionate distanc e (% mortality at dilution next above 50% – Mortality at at dilution next below or 71-50 21 —- = — = 0. 36 proportionate distance 71-13 58The dilution factor must also be considered, i. e. , 2-fold, 4-fold, 10-fold, etc. and the proportionate distance corrected (multiplied) by the log10 of the dilution factor (2-fold = 0. 3, 5-fold = 0. 7, 10-fold = 1, etc. ) The final estimate is determined by this formula: Negative log10 of LD50 end point = negative log of dilution above 50% mortality plus the proportionate distance factor (corrected for dilution series used) Negative log of dilution above 50% mortality –3. 00 Proportionate distance (0. 36) x dilution factor (log10-1= -1)= – 0. 36 Negative log LD50= -3. 36 LD50=10-3. 36 antilog of 10. 36=2. 29 LD50 titer -3. 36 =2. 29 x 103 / volume inoculated LD50 Calculation Inoculum: |DILUTION |DEAD |ALIVE |CUMULATIVE DEAD |CUUMULATIVE ALIVE |LD50= | |EXAMPLE |10-1 |6 |0 |17 |0 |(a-b)(c+d) | | | | | | | |2[(ax d)-(bxc)] | |Test System: |10-2 |6 |0 |11 |0 |=(3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | |Date Inoculated |10-3 |4 |2 |5 |2 |=24 =0. 36 | | | | | | | |66 | | |10-4 |1 |5 |1 |7 |LD50 = 3. 36 | | |10-5 |0 |6 |0 |13 | | |Inoculum |Dilution |Dead |Alive Cumulative Dead ( |Cumulative Alive ( | | | | | | | | |LD50= | |Test Systems: | | | | | |(a-b) (c+d) | | | | | | | |2((a x d) – (b x c)( | | | | | | | | | |Date Inoculated: | | | | | |= (3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | | | | | | | |=___________ | | | | | | | | | Inoculum |Dilution |Dead |Alive |Cumulative Dead ( |Cumulative Alive ( | | | | | | | | |LD50= | |Test Systems: | | | | | |(a-b) (c+d) | | | | | | | |2((a x d) – (b x c)( | | | | | | | | | |Date Inoculated: | | | | | |= (3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | | | | | | | |=___________ | | | | | | | | | 2. Spreaman-Karber Method Estimation of the 50% endpoint by the Spearman-Karber method is much simpler. The formula is: Negative log10 of LD50 = X – d (P-0. 5) here X = log10 of the highest concentration used (lowest dilution); d = log10 of the dilution factor, and p = sum of % mortality at each dilution100 Using the same data chart above the following number are obtained: d(10-1)(do-2)(10-3)(10-4) Neg log : LD50 = 1. 0 -1(100 + 100 + 66 + 17)-0. 5 100 = -1 [1(2. 84-0. 5)] = -1 2. 34 = -3. 34 LD50 antilog of 10. 34 = 2. 19 LD50 titer= 2. 19 x 103 volume inoculated Note that the two methods produce slightly different results using the same data. The Spearman-Karber method is considered to be the more accurate. The Spearman-Karber method can be simplified even more if signs are neglected and common sense used.This formula is: Neg log LD50 = X + d (P + 0. 5) Where X = log10 of highest dilution showing 100% mortality; d = log10 of dilution factor; p = proportion of positives above dilution X Xdp Neg. log LD50 = 2 + 1 (4/6) = 1/6 + 0. 5) = 2 + 1 (. 67 + . 17 + 0. 5) = 2 + 1. 34 = 3. 34 The appropriate sign can then be inserted: LD50 = 10-3. 34 These formulas can also be used to estimate 50% endpoints in neutralization tests. Here, absence of the predetermined criterion is counted and used for the calculations. III. SEROLOGIC TECHNIQUES A. Hemagglutination Many viruses or viral antigens are capable of specifically and non-covalently binding to receptors on the surface of red blood cells (RBCs).When the right volumes of these viruses and RBC’s are mixed, the viruses bridge the RBCs to form a lattice which settles out of suspension in a uniformly thin shield on the bottom of a test tube or conical well. This phenomenon was first described by Hirst in 1941 and is known as hemagglutination (HA). the HA titer of a virus can be determined by mixing serial dilutions of a virus with a constant amount of RBCs which are usually prepared as a 0. 25%-1. 0% suspension in physiological saline. the highest dilution which agglutinates the RBCs is the endpoint. The HA titer is the reciprocal of the endpo int dilution, and that dilution is said to contain one HA Unit (HAU) of virus in the original volume.Unagglutinated RBCs sediment to a packed disc (â€Å"button†) on the bottom of the test tube or well. The viruses known to cause hemagglutination are heterogeneous but can be grouped according to the nature of their hemagglutinating protein (hemagglutinin). The hemagglutinin on the virion of influenza and the paramyxoviruses is a glycoprotein. These viruses, but not any others, also carry an enzyme, neuraminidase, which destroys the glycolipid receptors on the RBC surface and allows the virus to elute (unless the HA is carried out at a temperature too low for the enzyme to act). Certain toga and Coxsackie viruses possess a hemagglutinin but do not possess a neuraminidase-like enzyme. astidious conditions are necessary for these viruses to hemagglutinate and usually cells from only a very few species can be can be used. Vaccinia virus has a lipoprotein hemagglutinin associated with a soluble fraction separable from the viral particle itself. Some viruses agglutinate RBCs from a limited number of course and some HA reactions require careful control of pH, temperature and ionic conditions (see Table 5-1, p. 100-101, Rovozzo & Burke). We will perform the HA test with a paramyxovirus which will agglutinate human type O, bovine, guinea pig or chicken RBCs over fairly broad ranges of pH (6. 0-8. 0) and temperature (4-25oC). Material needed: 1 cc syringes 0. 025 ml microtiter tips 0. 25 ml microdiluters Microtiter plates with 96-V-bottom wells Phosphate buffered saline (PBS) Paramyxovirus, in form of allantoic fluid harvested 48-96 hr after infection of 10-day embryonating eggs Washed RBCs, 0. 5% in PBS Go-no-go test papers Dilution tubes and 1 ml pipets *Use only the top half the microtiter plate. Procedure: 1. With the micropipet (a microtiter tip attached to a 1cc syringe) held vertically, dispense 0. 025 ml (1 drop) PBS each into columns 2 through 12 of rows A. B, C, and D of the microtiter plate. Also put 0. 025 ml PBS into wells 1 through 4 of row H for controls. 2. Make a 1:10 dilution of virus in PBS in a dilution tube.With the same pipet used to dispense PBS, put 0. 025 ml of the 1:10 virus dilution into each well of columns 1 and 2, rows A-D. 3. Test the delivery volume of the microdiluter by immersing the tip in PBS then touching to the center of a circle on the go-no-go paper. Now begin dilutions by immersing the dry diluter in well two, rotating to mix and pick up 0. 025 ml of fluid, and transferring to well three. Continue rotating and transferring through row 12. Two lines may be diluted simultaneously if desired. After removal of 0. 025 ml from row 12, dip the diluter in disinfectant, then distilled water, then flame. Do not flame with protein or salt in the diluter.Do not add virus to the controls. 4. With a new syringe and tip add 0. 025 ml (1 drop) 0. 5% RBCs (mix suspension well before pipetting) to every well, includin g controls. Mix well by running a hard object down the underside of the plate. 5. Allow to stand at room temperature until the controls and higher virus dilutions have RBCs settled into a â€Å"button† in the point of the V, and positive wells have RBCs uniformly spread over the entire bottom of the well. This will take 1-2 hr. Then refrigerate the plate. 6. Read the HA titer as the reciprocal of the dilution of the last well showing positive HA. Calculate the dilution which contains 4HAU/0. 25ml for use in the HI test.Be sure to check controls for spontaneous agglutination. B. Hemagglutination Inhibition Viral hemagglutination may be inhibited in several ways. By combining with viral antigens which normally interact with RBC receptors, specific anti-viral antibodies can prevent the virus cell interaction which normally brings about hemagglutination. Since infection with a virus will elicit production by the host animal of antibodies directed against each virus-induced protei n, including the hemagglutinin, inhibition of hemagglutination by an animal’s serum indicates that the animal has been infected by the virus. A high HI titer may indicate that the infection was recent.A four-fold rise in titer between two serum samples taken a few weeks apart (as during acute and convalescent phases of a disease) indicates that infection occurred during the period between the sampling times. If the viral hemagglutinin is also the protein by which the virus attaches to cells susceptible to infection, a high HI titer shows an animal to be immune to reinfection. HI is carried out in much the same way has HA. The serum is diluted in microtiter plates and each dilution is allowed to react with a constant dose of virus (usually four HAU) for an interval of 15 min to one hr before RBCs are added. The reciprocal of the highest serum dilution which inhibits HA is the HI titer.Several controls are necessary (1) The lowest dilution of serum used in the test must be incu bated alone with RBCs to determine if it contains heterophile antibodies which cause RVC agglutination. (2) The virus must be back titrated to see that the proper dose was added to the test wells. (3) A known non-immune serum from the same animal species must be titrated (usually before the HI test is performed) to see if it contains non-specific inhibitors. Heterophile antigens are a group of shared antigens with over-lapping specificities. They are found in some plants (corn, spinach) some microorganisms (Pneumococcus, E. coli), and some fish and animal tissues (carp, toad, guinea pig, horse, man etc. Heterophile antibodies against these antigens will cross react with cells and fluids from the above-listed species. In the case of RBCs as the heterophile antigen, if heterophile antibodies against them are present, hemagglutination will occur, possibly masking the presence of the hemagglutination-inhibition reaction caused by anti viral antibodies. Nonspecific inhibitors of hemagglu -tination may also be found in the serum of man and animals. Their nature differs for different viruses and even for different strains of viruses, such as influenza virus. Serum inhibitors also differ in different species. Inhibitors may be of low titer, or in some cases higher than the actual antibody titers, thus masking its diagnostic importance.Methods which have been used to remove inhibitors include: (1) Heating at 56-1/4 for 30 min, (2) treatment with receptor-destroying enzyme (neuraminidase), trypsin and/or periodate, (3) absorption with kaolin, (4) extraction with acetone, (5) precipitation of beta-lipoproteins with heparin and manganous chloride or with dextran sulphate and calcium chloride. No single method is universally applicable. Sometimes more than one method must be used. Antibody titers can be depressed by some of these procedures. The final control used is the RVC saline control to check for self-agglutinating RBC. Table 1. Example of HA and HI |Virus |Antigen So urce |RBC |Temperature |Non. Sp. Inhib.Removal | |Influenza A and B |CAS fluid or cell culture|Chicken, Human O |Room |Neuraminidase | | |fluid | | | | |Mumps |CAS fluid |Chicken |Room |Neuraminidase | |Coxsackie |Cells culture fluid |Fowl |Room |Kaolin | |Rubella |Cell culture fluid |one-day old chicken, goose |4oC |Heparin and Manganous chloride| |Adenoviruses |Cell culture fluid |Rat, rhesus monkey |37oC/room |Not required | Procedure 1.Add one drop (0. 025 ml) PBS diluent to all wells of the microtiter plate. 2. You will be given three serums. One will be untreated, one treated for removal of non-specific inhibitors and/or heterophile antibodies, and one known negative serum. Your results will be compiled with the class results to clarify the total experiment. using the microdiluters, add 0. 025 ml test serum to well A of row 1 and 2. Dilute out to well H. The first well is a 1/2 dilution of serum and row H is 1/256. Add 0. 025 ml of the same test serum to row 7, the serum contr ol well. Dilute to well H as before. Carefully rinse the diluters and repeat with test serum 2 and test serum 3, sing rows 3, 4, 8 and 5, 6, 9 respectively. 3. Add 0. 025 ml of the challenge virus with the microdiluters to well A of rows 10 and 11. Dilute to well H. This is the antigen (virus) back titration and control. The highest dilution with complete hemagglutination is 1 HA unit. 4. Using the same â€Å"micro-pipet† as in #1, add another drop of PBS to all wells of rows 7-12. Empty the pipet and refill with the hemagglutinin (virus). Add one drop hemagglutinin to all wells of rows 1-6. Mix well. 5. Incubate at room temperature 30-60 minutes. 6. Using a new pipet, add one drop 0. 5% bovine RBC to all wells. Mix well. Store at 4oC and read the next day.Antibody titers are the highest dilution that inhibits hemagglutination (forms a distinct button). C. Hemadsorption Certain enveloped hemagglutinating viruses cause the insertion of viral hemagglutinins into the plasma memb rane of cells in which they are replicating. These modified areas of the cell surface are the sites at which progeny virus particles will mature. If agglutinable RBCs are brought into contact with hemagglutinin-containing surfaces of cultured cells, the RBCs will specifically bind to the infected cells. This phenomenon, known as hemadsorption, is particularly useful in detecting infection by viruses which cause little morphological change in infected cells. Procedure 1.Pour off medium from a tube of cultured cells infected with an orthomyxovirus or a paramyxovirus and from a tube of uninfected cells. 2. Wash monolayer thoroughly but gently with two rinses of 3 ml of physiological saline. 3. Add 0. 2 to 0. 5 ml 0. 5% bovine RBCs in saline. Allow RBC suspension to cover cell layer. Incubate at room temperature for 10-15 minutes. 4. Pour off RVC suspension and wash 2x with 2-3 ml saline. 5. Examine under microscope. Infected cells should have entire surface covered with RBCs. Non speci fic binding will cover only a few sites per cultured cell. SERUM NEUTRALIZATION The neutralization test estimates the capacity of a specific serum antibody to neutralize a virus biological activity.Major uses for this test include the identification of unknown virus or antibody, the determination of antibody levels, the comparison of antigenically related viruses and the study of the kinetics of antigen-antibody reactions. Viruses and the study of the kinetics of antigen-antibody reactions. Neutralization can occur by several mechanisms. Virus adsorption to cells may be inhibited by alteration of the configuration of cell receptor sites or by prevention of viral attachment. Virus degradation may be enhanced by interference with post-engulfment stages of virus replication, by prevention of release of functional virus cores into the cytoplasm or by the degradation of virus-Ab complexes within phagosomes.Also, complement-mediated reactions may enhance neutralization by production of le sions in the viral envelope. Several factors must be considered when performing a neutralization assay. Sensitivity of the test is related to the degree of susceptibility of the indicator host system to infection with the virus. The neutralization reaction is readily reversible by dilution with saline, by ultrasonic treatment or by lowering pH. Finally, the time required to reach equilibrium may vary with different systems. When performing the neutralization test two systems are used. The reaction system is incubation of virus and specific antisera until equilibrium is reached. The indicator system is the inoculation of the virus-Ab mixture into a susceptible host.If neutralizing anti-bodies are not present lesions such as pocks or plaques will be seen in the host. If neutralizing antibodies are present there will be no lesions. There are two techniques commonly used for the neutralization test. In the alpha procedure a constant serum concentration is added to serial log dilutions o f virus. The mixture is incubated and inoculated into an appropriate host system. In the beta procedure a constant virus concentration is incubated in serial two-fold dilutions of serum before inoculation into the host. The beta procedure is most commonly used because of its sensitivity, ability to measure antibody titer and its economical use of serum.The alpha procedure is not as sensitive and may be more subject to non-specific inhibition. It is more frequently used for comparative studies. Alpha Neutralization test Materials Needed: Flat-bottomed MT plate with bovine cell monolayer MT transfer plate with lid and holder MT tips 1 cc syringes serum samples stock virus MEM diluent dilution tubes sterile distilled water in beaker Procedure: Use aseptic technique. a. Make serial 10-fold dilutions of stock virus to 10-8 using MEM and dilution tubes (0. 2 plus 1. 8 ml). b. Using sterile 1 cc syringe and microtip add 1 drop (0. 025 ml) diluent (MEM) to rows 7 and 8 wells A-H, and rows 9 , 10 and 11 wells A and B of the transfer plate. c. Using the same syringe and microtip add 0. 25 ml of the virus dilutions to rows 1-8 as follows: 10-8 into wells H, 10-7 into wells G, and so on finishing with 10-1 in wells A. d. Using a new syringe and microtip add 0. 025 ml test serum A to rows 1 and 2 wells A-H, and row 9 wells A and B. Rinse syringe and microtip with sterile water and add 0. 025 ml serum B to rows 3 and 4 wells A-H, and row 10 wells A and B. Again rinse out syringe and microtip with sterile water and add 0. 025 ml serum C to rows 5 and 6 wells A-H, and row 11 wells A and B. (Row 9, 10, and 11 are serum controls). e. Tissue culture controls are the unused portion of the plate. f. Incubate virus and serum at room temperature for 30 minutes, then transfer reagents to cell cultures. SerumVirusSerum Controls A B C 123456789101112 ABC A |10-1 |No | |B |10-2 |Virus | |C |10-3 | | |D |10-4 Virus | | |E |10-5 | | |F |10-6 | | |G |10-7 | | |H |10-8 | | Beta Neutral ization Test Materials Needed: Flat-bottomed MT plate with lid 1 cc syringes MT tips Mt diluters sterile distilled water in beaker MEM diluent serum samples virus, 25-50 TCID50 bovine cell suspension Procedure: Use aseptic technique. a. Add 1 drop (0. 025 ml) diluent (MEM) to rows 1-8 wells A-H. b.Make 2-fold dilutions of serum through row H (final dilution 1:256), cleaning microdiluters in sterile distilled water between serums. c. Using the same syringe and microtip as in step a, fill with pretitrated (25-50 TCID50) IBR virus and add 0. 025 ml to rows 1-4 wells A-H, and to rows 7 and 8 wells A. d. Using rinsed microdiluters make 2-fold dilutions of the virus in rows 7 and 8 wells A-H. e. Incubate at room temperature for 30 minutes. f. Add 2 drops (0. 05 ml) of bovine cell suspension using a new 1 cc syringe and microtip to all wells of the test plus a few extra for tissue culture controls. Controls Serum ASerum BABVirus 123456789101112 A |1:2 | | | |B |1:4 |No | | |C |1:8 |Virus | | |D |1:16 | | | |E |1:32 | | | |F |1:64 | | | |G |1:128 | | | |H |1:256 | | | IV. CELL CULTURE Because viruses are obligate intracellular parasites, they cannot replicate in any cell-free medium, and thus require living cells from a suitable host within which to multiply.Animals such as mice and embryonating avian eggs may be used for the propagation of viruses, but for various reasons (time, cost, ease of handling, etc. ) the propagation of most viruses in a cultural medium of living cells is the method of choice today. More than half a century has elapsed since animal cells were first grown in vitro. In 1912 Carrel began growing bits of chick heart in drops of horse plasma. The cells at the edge of the explant divided and grew out of the plasma clot. The explants died within a few days, and Careel reasoned that their death was due to exhaustion of nutrients. He found that cells from a given explant could be maintained indefinitely if they were periodically subdivided and fed with a sterile aqueous extract of whole chick embryos.In the early 1950’s, Earle developed a technique for dissociating cells from a whole chick embryo from each other with trypsin. When this suspension of single cells was mixed with plasma and embryo extract and placed in a sterile glass container, the cells adhered to the glass and divided to form a primary culture. The primary culture contained a variety of cell types including macrophages, muscle fibers, etc. The cells grew to a monolayer, a thin sheet of cells (one layer in thickness) which covered the entire bottom surface of their culture vessel, and then stopped dividing. The cells could then be redispersed with trypsin and planted in new culture vessels containing fresh media.These secondary cultures contained fewer cell types than did the primary cell cultures, as many of the differentiated primary cells were out-competed and did not survive the transfer. Often, secondary cultures are composed entirely of spind le-shaped cells called fibroblasts because of their similarity to cultured connective tissue. Cells derived from kidneys and from certain carcinomas have a polygonal appearance in culture. Because of their tissue of origin, they and other cells with similar morphology are call epithelial. Cells may be grown in vitro in several ways. Organ cultures, if carefully handled, maintain their original architecture and functions for several days or sometimes weeks.Slices of organs (which are actually tissue cultures) consisting of respiratory epithelium have been used to study the histopathogenesis of infection by respiratory viruses that can only be grown outside of their natural host by using organ cultures. The term tissue culture was original applied to explants of tissue embedded in plasma. the term subsequently became associated with the culture of cells in general and is now obsolete in its original sense. Cell culture is the term most widely used today. It refers to tissue dissociate d into a suspension of single cells, which after being washed and counted, are diluted in growth medium and allowed to settle on to the flat bottom surface of a specially treated plastic or glass container.Most types of cells adhere quickly, and under optimum conditions they will undergo mitosis about once a day until the surface is covered with a confluent cell monolayer. There are three main types of cultured cells. The difference in these types lies in the number of times the cells can divide. 1. Primary cell cultures When cells are taken freshly from animals and placed in culture, the cultures consist of a wide variety of cell types, most of which are capable of very limited growth in vitro, usually fewer than ten divisions. These cells retain their diploid karyotype, the chromosome number and morphology of their in vivo tissues of origin. They also retain some of the differentiated characteristics which they possessed in vivo. Because of this, these cells support the replicatio n of a wide range viruses.Primary cultures derived from monkey kidney and mouse and chick embryos are commonly used for diagnostic purposes and laboratory experiments. 2. Diploid cell strains. Some primary cells can be passed through secondary and several subsequent subcultures while retaining their original characteristics. After 20-50 passages in vitro, these diploid cell strains usually undergo a crisis in which their growth rate slows and they eventually die out. Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production. 3. Continuous cell lines. Certain cultured cells, notably mouse embryo fibroblasts and human carcinoma cells, are able to survive the growth crises and undergo indefinite propagation in vitro.After an initial slowing down, these continuous cell lines grow more rapidly than before, their karyotype becomes abnormal (aneuploid) and other poorly understood changes take place which make the cells immortal. The cells are now â€Å"dedifferentiated†, having lost the specialized morphology, and biochemical abilities they possessed as differentiated cells in vivo. Continuous cell lines such as KB and Hela, both derived from human others derived from mice (L929) and hamsters *BHK), are widely used in diagnostic and experimental virology. The development during World War II of antibiotics simplified long-term animal cell culture by minimizing the problems of bacterial and fungal contamination.Another important discovery was made by Eagle in the 1950’s when he determined the minimal nutritional requirements of cultured cells. He began by showing that Hela and Mouse L-cells would grow in a mixture of salts, amino acids, vitamins and cofactors, carbohydrates and horse serum. By eliminating one component at a time, he then determined which nutrients were essential for cell growth. His minimal essential medium (MEM) contains 13 amino acids (human tissue in vivo requires only 8), 8 vitamins and cofactors, glucose as any energy source and a physiological salt solution which is isotonic to the cell. The pH is maintained at 7. 2-7. 4 by NAHCO3 is equilibrium with CO2.The pH indicator phenol red is usually incorporated into the medium, which turns red-purple if the medium is alkaline, yellow if the medium is acidic, and remains red if the pH is suitable. Serum in concentrations of 1-10% must beaded to the medium to provide the cells with additional undefined factors, without which most cells will not grow. Most animal cells must be kept incubated at 37oC. If cells are grown in vessels open to the atmosphere, their incubator must be humidified and contain an increased CO2 concentration. Some nonvolatile phosphate or substituted sulfonic acid buffers (HEPES, TES) eliminate the requirement for incubators to be gassed with CO2. With the advent of cell culture, many animal viruses have been propagated in vitro, and hundreds of previously unknown viruses have been isol ated and identified.The discovery of the adenoviruses, echoviruses, and rhinoviruses, for example, is directly attributable to the use of cultured cells, as is the revolution in the diagnosis of viral diseases and the development of poliomyelitis, measles, and rubella vaccines. A. Culture of Primary Chick Embryo Fibroblasts (CEF) Materials 10-12 days old embryonated eggs Forceps and scissors Sterile petri dishes Sterile 250ml flask with magnetic bar Sterile 30 oz prescription bottles containing MEM & 5% lamb serum Sterile PBS Sterile 0. 5% trypsin (STV) Sterile 15ml centrifuge tubes containing 0. 5 ml serum Hemocytometers 1ml and 10ml pipets Sterile Dulbecco’s saline Procedure 1. Disinfect the surface of the egg over the air sac.With scissors or blunt end of forceps, break shell over air sac. Sterilize forceps by dipping in alcohol and flaming. Peel away shell over air sac, resterilize forceps and pull back shell membrane and chorioallantoic membrane to expose embryo. 2. Rest erilize forceps, grasp embryo loosely around neck, and remove from egg to sterile petri dish. 3. Using two forceps, or scissors plus forceps, decapitate and eviscerate embryo. Mince remainder of embryo to very small fragments. 4. Add about 10ml sterile Dulbecco’s saline to tissue fragments in petri dish, swirl to suspend fragments, and carefully pour into 250ml flask. With flask covered, continue swirling for 2-3 min. to wash tissue fragments.Tilt flask, allow fragments to settle, and gently decant saline. 5. Add 12ml sterile trypsin to fragments in flask, cover, and stir with magnetic bar for 15 min. Tilt flask, allow fragments to settle, and pour trypsin cell suspension into 15ml centrifuge tube containing 1ml serum. The serum contains a trypsin inhibitor which will prevent further damage to cell membranes but he enzyme (note: it is preferable to treat the tissue with multiple short applications of trypsin rather than a few long ones, in order to minimize enzymatic damage t o cell membranes. However, limitations of time require us to use the shorter method. ) 6. Add 12ml sterile trypsin to fragments and repeat step 5.At the end of this second treatment, size of tissue fragments would be greatly reduced and a large number of single cells should be suspended in trypsin. 7. Balance centrifuge tubes against one another and centrifuge at 1500 rpm for 10 min. Carefully decant off supernatant and resuspend pooled cell pellets in 1ml MEM. Make a 1:10 dilution of the cell suspension in MEM for counting in a hemocytometer. 8. In most hemocytometers each heavily etched square in 1mm on each side. The depth of the chamber is 0. 1mm. Count the cells in 0. 13 mm and calculate the number of cells in your original suspension. Dilute to give 8ml with 2-8 x 105 cells/ml in MEM, place in prescription bottle, replace cap tightly, and incubate on flat side at 37oC. 9. Be sure to examine cells periodically.Actively growing cells produce acidic metabolic by-products, and thu s the pH of the medium may need to be adjusted by the addition of a few drops of 7. 5% NAHCO3. If floating (dead) cells are present the medium may need to be changed. B. TRANSFER OF CELL CULTURES After cultured cells have formed a confluent monolayer on the surface of their culture vessel, they may be removed from the surface, diluted, and seeded into new vessels. If the initial culture was primary, the new cultures are called secondary, and are likely to consist of fewer cell types. Removal of cells from glass surfaces may be by either physical methods – scraping with a sterile rubber policeman – or chemical methods – proteolytic enzymes or chelating agents – or a combination of the two.After removal, cells are pipetted up and down and diluted appropriately in fresh secondary culturing, and after one becomes familiar with the growth characteristics of a certain cell types, counting can usually be dispensed with. We will transfer a cell line of bovine cel ls by use of a mixture of trypsin and EDTA (versene) in physiological saline (STV = saline, trypsin, versene): 1. Pour off the medium from a 3 oz. prescription bottle containing a confluent cell monolayer. 2. Wash the monolayer with 5-10 ml of physiological saline (Saline A) rinse well without shaking (shaking produces bubbles) and pour off. 3. Add 0. 5 ml STV to the bottle and incubate, with STV covering cells, at 37oC for 2-15 min.Observe periodically to determine when cells are loosened from glass (note: STV will contain a pH indicator and should have a pH of 7. 0-8. 0. Below pH 7. 0, trypsin is inactive. A pH above 8. 0 is damaging to cells. ) 4. When cells are seen to detach from glass upon shaking, add 6 ml fresh medium and suspend cells by pipetting up and down a few times. 5. Add 10ml more medium and mix to get even cell suspension. 6. Seed 1 ml cell suspension in to each of 8 culture tubes, stopper tightly, and incubate in rack which holds tubes at slight angle from horizon tal. Seed remaining 8 ml cell suspension into a new 3 oz. prescription bottle or a 25 cm2 plastic flask. C.PRESERVATION OFCULTURED CELLS BYFREEZING Viability of viruses and bacteria is preserved during freezing, but originally attempts to preserve animal cells by freezing resulted in cell death. This was first thought to be due to laceration of cell plasma membranes by ice crystals, but more recent evidence suggests the cause may be osmotic changes during freezing which give rise to irreversible changes in lipoprotein complexes in intracellular membranes. In any event, the answer to animal cell preservation has proved to be addition of glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO) to the medium and slow freezing, ideally at a cooling rate of one centigrade degree per minute.Cells must be stored at 70oC or lower (ideally in liquid N2 at 196oC), and when they are recovered, thawing must be rapid. With careful technique, 50-80% of the cells of a healthy culture will survive f reezing. Procedure 1. Remove confluent cell monolayer from culture vessel by method described in cell transfer procedure. After centrifugation, resuspend cells in 1 ml medium containing 15% serum and 7. 5% DMSO and placed in small snap-top tube. 2. Immediately place tubes in an ice bath. They will then be transferred to a styrofoam container and refrigerated. After 20-30 min, when cells have dropped to 4o, they will be transferred to a 20o freezer for 20-30 min, then to the 70o freezer for storage.Alternatively, the tubes can be placed in cotton-or polystyrene-insulated containers and placed directly in the 70o freezer for slow cooling. If cells are to be stored in liquid N2, they must be placed in sealed ampoules. 3. To recover, cells, remove tubes from 70o and place directly in 37o water bath. When thawing is barely complete, add contents of tube to a 25 cm2 flask containing 15 ml MEM + 10% fetal calf serum. Culture medium will be changed for your approximately 4 hrs. later (after cells have attached) to reduce the toxicity of DMSO for cells at 37oC. D. Effect of Viral Infection on the Host Cell During the time that synthesis of viral components is occurring in the infected cell, the cell undergoes characteristic changes.These changes are usually observed in tissue culture where infection of cells is more easily synchronized and where the cells can be observed frequently during the course of infection. Morphological changes in cells caused by viral infection are called cytopathic effects (CPE): the responsible virus is said to be cytopathogenic. The degree of visible damage to cells caused by viral infection varies greatly. Some viruses cause very little or no CPE. Their presence can be detected only by hemadsorption (already discussed) or interference, in which infected cell cultures showing no CPE inhibit the replication of another virus subsequently introduced into the cultures.On the other hand, some viruses cause a complete and rapid destruction of the cell monolayer after infection. The histological appearance of the CPE caused by some of these cytocidal viruses may be sufficiently characteristic to allow provisional identification of the virus. Some CPE can be readily observed in unfixed, unstained cells, under low power of the light microscope, with the condenser down and the iris diaphragm partly closed to obtain the contrast needed for viewing translucent cells. Several types of CPE are distinguishable in living cultures, but fixation and staining of the cells is necessary to see such manifestations of viral infection as inclusion bodies and syncytia.Recognizing CPE and using it as a diagnostic tool requires much experience in examining both stained and unstained cultures of many cell types. Listed below are several general types of CPE. Keep in mind that a given virus may not conform to the norm for its family, or it may produce different CPE in different host cell types. The best knowledge of viral CPE comes from experience . 1. Total destruction of the cell monolayer is the most severe form of CPE. All cells in the monolayer rapidly shrink and become dense (Pyknosis) and detach from the glass within 72 hours. This CPE is typical of most enteroviruses. 2. Sub-total destruction consists of detachment (death) of some but not all of the cells in the monolayer.The alpha-togaviruses, some picorna viruses, and some of the paramyxoviruses may cause this type of CPE. 3. Focal degeneration is characteristic of the herpesviruses and poxviruses. Instead of causing a generalized destruction of the cell monolayer, these viruses produce localized areas (foci) of infection. The focal nature of these lesions is due to direct cell-to-cell transfer of virus rather than diffusion through the extra-cellular medium. Cells initially become enlarged, rounded, refractile (more easily seen), and eventually detach from the glass, leaving cleared areas surrounded by rounded up cells as the infection spreads concentrically. Stran ding of the cytoplasm is usually pronounced and cell fusion may be evident. 4.Swelling and clumping of cells before detachment is typical of adenoviruses. Infected cells greatly enlarge and clump together in â€Å"grape-like† clusters. 5. Foamy degeneration (vocuolization) is due to the production of large and/or numerous cytoplasmic vacuole. Several virus families including certain retroviruses, paramyxoviruses, and togaviruses may cause vocuolization. 6. Cell fusion (syncytium or polykaryon formation) involves the fusion of the plasma membranes of 4 or more cells to produce one enlarged cell with 4 or more nuclei. Polykaryon formation may be the only detectable CPE of some paramyxoviruses; herpesviruses may also produce syncytia. 7. Inclusion bodies are areas of altered staining in cells.Depending on the causative virus, these inclusions may be single or multiple, large or small, round or irregularly shaped, intranuclear or intracytoplasmic, eosinophilic (pink staining) or basophilic (blue-purple staining). In most cases they represent areas of the cell where viral protein or nucleic acid is being synthesized or where virions are being assembled, but in some cases no virus is present and the inclusion bodies represent areas of viral scarring. V. BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF VIRUSES There are many biochemical and biophysical tests which can be used for classification of viruses. We will perform four of these test using â€Å"unknown† viruses: viral sensitivity to lipid solvents, determination of virus size, determination of virus nucleic acid type, and viral sensitivity pH and heat.The chart on p. 127 of your lab book may help in the identification of your virus. A. Viral Sensitivity to Lipid Solvents. The lipid sensitivity test is one of the most basic tests for characterization of viruses. There is a correlation between the presence of an envelope and the susceptibility of viruses to lipid solvents such as ether, chloroform, and detergents. Enveloped viruses require their lipid membrane for infectivity; because the test measures destruction of viral infectivity vs. untreated viral controls, it is an indirect test. All lipid coated viruses are sensitive to chloroform, whereas all but a few poxviruses are sensitive to ether.This is because the lipid components of the poxviruses are much diffe