Body Image in Brazil and Usa

Body Image in Brazil and USA Four thousand years ago the last of the mammoths were roaming the earth before extinction, anesthesia was still 3800 years away from being discovered, and tools were still being made out of stone. What else was happening that long ago? Humans were performing the first known cases of reconstructive and cosmetic surgeries, documenting back to skin grafts in ancient India. Between the first documented procedures and the early 1800's not a lot progressed aside from the basic tools being used. In 1827, Dr.John Peter Mettaue performed the first cleft palate operation using tools of his own design kicking off the modern plastic surgery advancements. Using the advancements in reconstructive surgeries became increasingly popular during World War I as it was used to save many soldiers' lives throughout the world. In the late 1940’s, following the second World War, the focus of plastic surgery began to shift from medical procedures to save lives in the milita ry to a more public and socialized practice. A boom in the 1960's spread rapidly after the introduction of silicone implants by Dr. Thomas Cronin.Sports Illustrated Magazine issued its first swim suit edition in 1964 featuring a five page spread of bikini clad perfect model bodies that the public was pressured to imitate. Despite the American involvement in Vietnam during the late 60's the trend continued to increase into the 70's when plastic surgery hit an all-time high due to the public discovery of its uses on all parts of the body. Over the decades, countries throughout the world including Brazil and The United States have adopted plastic surgery as an active part of their cultures despite monetary and health costs all because of media and social pressures.The United States and Brazil rank first and second in the world of most plastic surgery procedures, respectively. According to Dr. Daniela Dorneles de Andrade, a psychological research associate at the University of Vienna, t he United States alone underwent 30. 1 million cosmetic surgeries in the year 2009, enough cosmetic surgeries for one in every ten Americans to have undergone some sort of altering procedure. The United States is the only country to top the next leading country, Brazil, which reported 13. 7 million procedures.Based on its population, that amounts to one in every fifteen Brazilians volunteering for of these surgeries in the same year (Dorneles 75). The American Society of Plastic Surgeons reports the average cost of plastic surgery procedures being at five thousand dollars in the United States incurring a total revenue topping one hundred and fifty billion dollars a year from voluntary surgeries alone. The figure dwarfs Brazil's reported income from the same procedures however, topping just over fifty million dollars (ASPS).Professor Alexander Edmonds, of Macquarie University, reports that the lack of funds reported by Brazil is due to a philosophy that â€Å"the poor have a right t o be beautiful† (Edmonds, â€Å"Poor† 363). The thought that everyone has a right to undergo plastic surgery, even if they cannot afford it, has been adopted by many Brazilian surgeons. Brazilian surgeons have started clinics that are being funded by federal and municipal budgets to provide procedures to everyone regardless of economic ability (Edmonds, â€Å"Poor† 365). Such acts are not only costing people in American and Brazilian cultures money but also costing them their health.Both psychological and physical health are being put into jeopardy by the procedures themselves and also by the desire to have them done (Edmonds, â€Å"Learning† 470). Health care related spending has nearly tripled in the past three centuries, seventy-eight percent of which linked to complications of cosmetic surgery. Whether it is leaking silicone implants or infections, the surgeries that people are seeking out to make themselves more perfect on the outside are in fact lead ing to more problems than with what they started with (Dorneles 77).Why are people of the world putting themselves through these extensive procedures? University of Amsterdam professor, Alexander Edmonds, says it amounts to nothing more than acceptance and expectance. The pressure to appear as perfect as possible on the outside is largely placed upon the women in both Brazilian and American culture. Such pressure is put upon women, young women most heavily, by media and social groups alike. Social groups are driven by what they see in magazine or on television ads. Media thrives on what social groups are deeming appropriate amongst themselves.The vicious cycle of perfection that American teens and young adults face every day is the same pressure that is seen in Brazil. More and more young people are turning to evasive procedures to correct themselves every day. In 2010 the second most popular gift given to high school graduates in America, trailing closely behind a new car, was that of breast augmentations (Kreimer). These gifts are giving by family members or people who care about the young person's life and they feel that their child will thrive better in life if they help them achieve a better body.This thinking is passed on from generation to the next and is rapidly increasing. One teen who received such a gift was quoted saying, â€Å"My mother, grandmother, two aunts, and stepmother have implants, so if my mom is willing to pay for it, why not? † (qtd in Kriemer). The pressure to appear a certain way is becoming even more important to people of the world with no consideration for the health and financial implications. It is becoming accepted by cultures around the globe as a normal practice.If the past is any indication for the future this issue will become an uncontrollable epidemic. Something needs to be done about how media portrays people but are the thoughts of societies and morals of cultures being influenced by the media or is the media bei ng conformed by the cultures and societies serves? Works Cited ASPS (American Society of Plastic Surgeons). The Plastic Surgery Foundation, 2012. Web. 23 Oct. 2012. Dorneles de Andrade, Daniela. â€Å"On Norms and Bodies: Findings from Field Research on Cosmetic Surgery in Rio de Janeiro, Brazil. Reproductive Health Matters 18. 35 (2010) : 74-83. Print. Edmonds, Alexander. â€Å"Learning to Love Yourself: Esthetics, Health, and Therapeutics in Brazilian Plastic Surgery. † Routledge Journals 74. 4 (2009) : 465-489. Print. Edmonds, Alexander. â€Å"’The Poor Have the Right To Be Beautiful’: Cosmetic Surgery in Neoliberal Brazil. † Journal of the Royal Anthropological Institute 13. 1 (2007) : 363-381. Print. Kreimer, Susan. â€Å"Teens Getting Breast Implants for Graduation. † Womensenews. Women’s eNews Inc. , 6 June. 2004. Web. 26 Oct 2012.

A Dolls House

You have, perhaps, never heard a discussion or symposium on the subject, â€Å"Men-their role in the society. The discussion is always about women. Men have, perhaps, no role!Men can as well do, some of the jobs normally done by women. Even today, the best chefs are men! The never-ending talk to give equal rights to women has been going on since time immemorial. But all the Acts of Parliaments all over the world will not bring equality to the female gender.he change has to be achieved within-both men and women! How can equal rights to women be given? God has created her, given her the status of more-equal. Nobody can take that right away.It is the mother, who gives protection during the initial nine months to the divine creative force of the future-male or female! A female child is victimized at every step of life, from the moment of birth, and she sacrifices at every stage.   Women need to be the legal and spiritual equals of men!The three Act play by Henrik Ibsen is dominated by the main character Nora. The Doll’s House is symbolic—how Nora has been living within the specified rules applicable to housewife-submission, submission and more submission to the wishes of her husband! Before marriage, she submitted to her parents (mostly to the wishes of her father); now the place of submission is changed, but the procedure of male-dominance is the same-God bless her!With no scope to develop her own personality she is obliged to live under the ‘protective’ umbrella of her husband but not for long! Nora turns to be a dynamic rebel, gives up the structure that is considered sacred by the society and seeks the path of total transformation and rehabilitation of her own personality. What is marriage?Two distinct individuals, two separate personalities, born, bred and brought up in two different set of circumstance, try to come together from the day of marriage( if not courting earlier!) to find a common identity, a common goal and to be prec ise a common all! But soon the affected one finds out that idealism and the reality are poles apart!Why the scale of social justice tilts heavily against the womenfolk and their individual liberties are at a discount? Why one finds Nora in every family in one form or the other even during the 21st century? How long need the woman contain her suppressed emotions and move forward   to establish her own identity?It is the same old story through the Ages—it is between Him and Her! The family is run as per the dictates of Torvald, though his commands are honey-tongued. An attitude of ‘I know everything’ and ‘you know nothing’ is evident in his relationship with Nora.The sweet nothings he addresses to his wife like, â€Å"Is my little lark twittering there?†(Ibsen, 2007, p. 6) â€Å"Is it my little squirrel bustling about?†(p. 6) â€Å"Has my little spendthrift been wasting money again?†(p. 6) â€Å"You extravagant little personâ₠¬ ¦Ã¢â‚¬  (p.8) â€Å"Hasn’t Miss Sweet-Tooth been breaking rules in town today?†(p.10) are all indicators of his superiority complex. And Torvald very clearly issues his edict as for planning the family budget. He says, â€Å"That is like a woman! But seriously, Nora you know what I think about that. No debt, no borrowing.There can be no freedom or beauty about a home life that depends on borrowing and debt. We two have kept bravely on the straight road so far, and we will go on the same way for the short time longer that there need be any struggle.† (p.7) Ibsen has tackled several themes, mixed them very well, like a woman’s illusion in marriage, wealth, feminism, betrayal, etc.How A Doll’s House has been created within the house, how the woman functions inside the demarcated boundaries—such issues are dealt with intelligently. In the later part of the play Nora walks away from her family. Her undoing and the reason for falling from the à ¢â‚¬Ëœgrace’ of her husband is– she forged a bank note to save her husband’s life when no one else could. How she is compelled to tell a series of lies, and it looks as though such behavior is part of her personality. She does not feel guilty about it but for Torvald it is a major and fundamental issue.â€Å"It is better to have the world united than the world divided; but it is better to have the world divided than the world destroyed,† said Mr. Winston Churchill at the time of II World War. Similarly instead of destroying the family and the psyche of children, by endless misunderstandings, honorable separation is better!Amin Maalouf in his book, â€Å"Leo the African†, describes a poignant situation of the woman in the medieval society   thus: â€Å"Gaudy Sarah   came to see me†¦to sell amulets, bracelets, perfumes made from lemon, ambergris, jasmine and water lilies, and to tell fortunes†¦.without lifting her eyes, she said these words, which I remember to this day.‘For us, the women of Granada, freedom is a deceitful form of bondage, and slavery a subtle from of freedom. Then saying no more, she took out a tiny greenish stopper bottle from her wicker basket.‘Tonight, you must pour three drops of this elixir in to a glass of orgeat syrup, and offer it to your husband with your own hand. He will come to you like a butterfly towards the light. Do it again after three nights, and again after seven.†(Maalouf, 1994, p.6) For the sake of getting the attention of her husband, what abnormal steps the woman is compelled to take!Conclusion:Norwegian playwright Henrik Ibsen was born in 1828. The play was written in 1879. It was performed in London in 1889.Nearly thirteen decades after the play is written, the issues touched upon and discussed in the play are like the counseling sessions for the female gender, in today’s materialistic civilization, highly impacted by the industrial and internet revolutions.Women have taken great strides and have already caught up with men in several important areas. This need not be construed as the race between the male and female genders—rather it is the reality! Equality is the birthright of one and all.

Free Trade and Protection Essay Example | Topics and Well Written Essays - 750 words

Free Trade and Protection - Essay Example There is no doubt pertaining to the fact that protection and free trade are the two approaches which can as much be resorted to by the developing world as by the developed world to meet their growth objectives and the domestic challenges. Especially more so when the US has an elaborate history of being open to the industrial products from foreign nations while strictly controlling the influx of their agricultural products. Pragmatically speaking, an allegiance of the developing world to the free trade will not be of any use to it, unless it aids in its objectives of poverty alleviation and extending health, nutrition, and education to the poorest of the poor. Considering the fact that large parts of China and India are facing a situation of severe drought, the producers of critical agricultural products in these two nations definitely needs to be protected. With farmers in the Telangana (India’s cotton belt) and Vidarbha (India’s breadbasket) parts of India committing s uicide owing to the massive losses incurred by them on a continuous basis and the inflation in India soaring to double-digit figures, India’s farming sector certainly needs to be protected from the onslaught of American farm products. The gravity of this tragedy is further accentuated by the fact that loan waivers of INR 60, 00 million extended by the Indian government failed to rescue the Indian farmers from a predominant mood of despondency, hopelessness, and frustration and the suicides are still continuing.... Especially more so when the US has an elaborate history of being open to the industrial products from foreign nations while strictly controlling the influx of their agricultural products. Pragmatically speaking, an allegiance of the developing world to the free trade will not be of any use to it, unless it aids in its objectives of poverty alleviation and extending health, nutrition and education to the poorest of the poor.Considering the fact that large parts of China and India are facing a situation of severe drought, the producers of critical agricultural products in these two nations definitely needs to be protected. With farmers in the Telengana (India's cotton belt) and Vidarbha (India's bread basket) parts of India committing suicide owing to the massive losses incurred by them on a continuous basis and the inflation in India soaring to double digit figures, India's farming sector certainly needs to be protected from the onslaught of American farm products. The gravity of this tragedy is further accentuated by the fact that a loan waver of INR 60, 00 million extended by the Indian government failed to rescue the Indian farmers from a predominant mood of despondency, hopelessness and frustration and the suicides are still continuing.Perhaps it is easy for the US to sing the songs of free trade, when its farming sector is primarily mechanized and employs merely 1.9 percent of its population. Contrary to this, China has nearly 23 percent of its population employed in agriculture while in India this figure stands at an astonishing 58 percent. Considering the contemporary recessionary global trends, the economic growth in India has already plummeted to 5.3 percent from the projected target of 7 percent. Large scale lay offs already becoming

Transportation by Sea Assignment Example | Topics and Well Written Essays - 3000 words

Transportation by Sea - Assignment Example A busy seaport should therefore be viewed as a powerful yardstick for measuring the development in the region in particular and in the country in general. But, the overall development of a port depends on several factors including that of having the port in a geographically advantageous location. The locational advantage, among other factors, plays a prominent role in the growth prospects of a port as it would naturally make the port accessible to the shipping lines of several countries. Apart from the locational advantage, a port should internally posses certain technical advantages too for efficient handling of port operations. and strictly prefer seaports that would reduce transportation costs and time. Port location is therefore one of the prime determinants of its development. The growth of a port also depends on the depth of water, warehousing facilities and the total area. These requirements, along with the importance of location, should be properly studied for port designing and construction depending on the type, size, number and frequency of vessels that would call at the port, type and volume of cargo that would be handled and needs of the warehousing facilities and terminals. ... agoon, in a deep natural bay or river would naturally attract the attention of shipping lines and develop very fast (Internet, Port Planning, Factors influencing sea port locations). Locational advantage When a port is located in a geographically convenient location joining several shipping routes and providing accessibility to several countries, it undoubtedly attains the geographically locational advantage. Such a superior port conspicuously draws the attention of the shipping lines and attracts huge demand for its services. If the port is also located in a deep water zone, it would facilitate the docking of the bigger ships (Internet, A new port in Shanghai, 20 miles out to sea, Para 9). Because of the innumerable advantages of having deep water ports, several countries, including India, have drawn up big plans in this regard (Internet, Ambitious growth plans for Kakinada deep water port, 3 2001). The main advantage of such a port is that its services are generally useful not only for the country but also for its neighbouring countries. Different countries, operating their shipping lines for the transportation of their different commodities and goods, would crave for utilising the facilities of such a port to meet their growing demands. When the advantageous location is effectively aided by the presence of industrial growth in the region, it adds magnificently to the port development (Internet, The fundamentals of ports management, function and role of an international port). Infrastructure While the locational advantage is the nature's gift for a port and serves as the basic ingredient for its development, the port location should be fortified by the presence of excellent infrastructural facilities to render quality services at reduced costs to various

The ableist conflation by Joel Reynolds Essay Example | Topics and Well Written Essays - 500 words

The ableist conflation by Joel Reynolds - Essay Example As many groups have done, disabled people have been engaged in what Anspach calls â€Å"identity politics†; politics that endeavor not only to change society’s conception and response to disabled people, but also to change the self-concepts of disabled people themselves. The work of disabled people in changing how they think about themselves continues, as the disability community struggles for self-definition and self-determination, as well as for civil rights. One of the most important problems facing the political struggle of people with disabilities is the necessity of developing a positive sense of identity. The very idea of a positive disability identity flies in the face of long-standing social â€Å"wisdom† about disability. The reason for this lies in the cultural beliefs about disability that have determined the status and perceptions of disabled people in our society today. Historically, disabled people were viewed as social and moral deviants, violations of the natural and cosmic order of the universe (Reynold, J). The response to such â€Å"deviance† was to protect society by separating disabled people from society in asylums, jails, basements, attics, etc. Disabled people were not considered fully human, had no role in society, and no basis for a positive social identity The theories of psychosocial identity development in onset disability, particularly minority identity development, provide a useful framework for exploring the concept of disability identity development as a minority culture phenomenon. Within the literature of identity development, few theorists have explicitly addressed the issue of disability, yet the models provide fertile ground for exploration. In contrast, an extensive body of rehabilitation literature exists that is grounded in the medical view of disability that has generated research findings that support and reinforce a medical

Innovation, Creativity and Enterprise in Markets Essay - 4

Innovation, Creativity and Enterprise in Markets - Essay Example The innovations that have been taking place in the company since its embarking upon a long-term journey have been illuminated during the due course of the report. The main reasons because of which Unilever opted to buy Ben & Jerry’s have also been discussed. Apart from that, the entrepreneurial aspect of this company has been explained and how entrepreneurship has played a practical role in the success of their business has been elucidated. All these elements have combined together to translate into profitable growth of their company along with its brand and have led it to its present state. The purpose of this report is to conduct a research on an organization and discuss in detail, how its presence does creates opportunities and prospects for its stakeholders in light of the theory of the same. Apart from that, the method by which the company innovates its processes and products in order to meet the ever changing needs of the consumers is also conversed. Furthermore, this report also concerns how the selected organization incorporates entrepreneurial principles in order to exploit the available opportunities in the market and undertake some level of risk-taking in order to derive the most benefits from the untapped market segments (Drucker, 1986). For this research, Ben & Jerry’s has been selected as it has been coming up with innovations in its flavors ever since it has come into being in 1978. The Ben & Jerry’s is an ice-cream, frozen yogurt and sorbet manufacturing company which was set up in 1978 as an ice-cream parlor on a renovated gas station in Vermont USA, by two entrepreneurs namely Ben Cohen and Jerry Greenfield with an investment of $12,000. The ice cream parlor soon gained popularity around the neighborhood and the pair began to pack their product and started to supply it to Mom and Pop stores and convenience stores in their Volkswagen Campervan. They eventually grew

Gaz de France Essay Example | Topics and Well Written Essays - 1000 words

Gaz de France - Essay Example The interest rate also plays an important role. The prevalent interest of FF against USD will influence FF-USD forward exchange rate. The government budget also plays a role in the exchange market; a deficit in the budget increases foreign currency demand. Among listed parameters; account balance, foreign exchange reserves and government budget are considered as the core parameters. the period from 1980 to the 1st quarter of 1986. Both account balance, and foreign reserves decreased during years from 1980 to 1982. It would depreciate FF against USD, which is also confirmed by the increase in the deficit of the government budget. During the years from 1982 through 1985, an increase in the current account and foreign reserves were observed; however, the budget deficit during the same time kept increasing. This trend would further depreciate the French Franc. Budget deficit considerably decreased in the 1st quarter of 1986 with respect to 1985; this will appreciate the Franc. A combined analysis of trend of account balance, foreign reserve and budget deficit explains that depreciation rate will be substantial during the years from 1980 through 1984, which will slow down in 1985 demonstrating further appreciation of FF in the subsequent year with respect to the previous years. The exchange rate between two currencies is influenced by various parameters. This assignment has selected three parameters; current account, foreign exchange reserves and government budget. The set forth above section presented a forecast of the behavior of the FF-USD exchange rate. The attached graph displays the movement of the real exchange rate for years from 1980 to the 1st quarter of 1986, which confirms the forecasted behavior. The FF depreciated from 1980 to 1982 because all three parameters; budget, balance and reserve show decrease in values from 1980 through 1982. The current balance and reserves showed an in increase from 1982 to 1985, but the budget deficit showed

PED 131 Essay Example | Topics and Well Written Essays - 500 words

PED 131 - Essay Example In the paper I got the second question wrong. The question was -When the command Cease Fire is given other than the range office. My answer to this was (b) Stop shooting immediately, lay the gun down on the bench and await further instructions. The reason I chose this answer was because when someone gives the command to cease fire it could be because there is some hazardous situation. Thus the best way to avoid any danger would be to stop firing and lay the gun down. But the correct answer is (d) since the correct thing to do would be to stop firing, point the gun in a safe direction and call or wait for the range officer to give further orders and if needed clarify as to why someone else gave the order. My answer to question 3 was because I felt that after notifying the range officer the best thing to do would be to open the action and remove the cartridge since in order to continue firing I would have to remove the faulty cartridge. But the correct answer to this is (d) since it is the range officer who is responsible to over see the removal of the cartridge. My answer to question 5 was (b) since in all the guns I have seen till date the safety switch has always been on the side and no where else.

Hospital improves patient case with data warehouse Study

Hospital improves patient with data warehouse - Case Study Example What this paper aims to achieve is develop an understanding of various economic theories in order to be able to explain the readers about various advancements being made in the world economy. Let’s start off by taking one side of the coin and discussing it in detail before moving on to the other one (Dean). The theory of Realism concentrates on shedding light on the way various roles played by various States around the globe in determining the trend of the international political economy. Moreover, it also focuses on the relation between the level of power possessed by a State and/or a group and how it affects the international economic trend. The supporters of Realism are often referred to as mercantilists or economic nationalists. What’s rather interesting about this school of thought is the fact that they believe that States across the globe find their motivation as the level of power they possess gets maximized. They further believe that power maximization is achieved through the help of international trade, and in this regard international trade policies are one of the many tools that can be put to use by States. Economic analysts who are supporters of Realism are of the view that the global economy works on terms which are majorly based on the interests and motives of powe rful States existing world over (Jonathan and Wight). The second theory that we are focusing upon here is the Marxist theory. This theory was invented by Karl Marx along with his friend and fellow co-author, Fredrick Angles. As mentioned earlier this theory mainly focuses upon differences in various social classes and motives of different workers. Karl Marx, or rather the Marxism theory seems to argue upon the fact that the existing conflict between the workers and owners of capital could only be amicably resolved provided the working class seize their power. Marxism theory

Endocrine disrupting compounds and human fertility Essay Example for Free

Endocrine disrupting compounds and human fertility Essay In the past 100 years, humans have introduced several hundreds of new compounds into the environment, which actually have affected the physiology of both plants and animals including humans (Propper, 2005). In most cases these deleterious effects are unintended and it was not predicted before that these compounds could have such effects on organisms. Therefore the actual mechanisms by which these compounds affect physiological functions of other organisms are not yet properly researched. When such compounds affect the endocrine systems they are called endocrine disrupting compounds. These compounds would affect different hormonal pathways and physiological functions such as reproduction, development, metabolism and even the behavior of humans and other animals. The present essay is intended to identify some of the endocrine disrupting compounds that affect human fertility, the mechanism of affecting, to analyze the weight of different evidences available and to analyze the current investigation techniques. Endocrine disrupting compounds have been defined as an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body which are responsible for the maintenance of homeostasis, reproduction, development and/or behavior (Kavloc et al. , 1996). The United States Environmental Protection Agency (USEPA) has accepted this definition as the most appropriate one in the year 2004. These compounds are capable of interfering with normal signaling mechanisms of the endocrine system. Either they could block or make changes in the synthesis of hormones, or they could mimic some of the endocrine compounds, thereby affecting the target organs. They could also affect the release of these hormones from the concerned glands and its transportation. They could also bind with the specific molecules to which hormone binds. These compounds are usually seen in pesticides, industrial effluents, pharmaceutical compounds, etc. Heavy metals also could induce endocrine disruption. Wastewater effluents from cities as well as from agricultural fields are sources of such compounds. The neuro- endocrine system might also get affected by these compounds causing changes in the reproductive organs and associated behaviors in humans. Most of the researches in this filed are concentrated towards the effect of these compounds on estrogen and other steroids responsible for reproduction (Propper, 2005). According to Caserto et al. (2008) these compounds could affect human heath seriously even when present in very small amounts. This is especially because many such chemicals would be these affecting a single target. There are many studies which reveal that waste water discharge in to natural waters have resulted in the changes in reproductive organs of aquatic fauna. This is because of the presence of 17 beta estradiol, estrogens, androgens, etc, in wastewater. These compounds are highly stable and therefore could not be removed completely from wastewater by various treatment procedures to reclaim the water. Traces of these compounds would be present in the drinking water, which is prepared from these natural waters into which the wastewater has been disposed. Bioaccumulation of these compounds in humans is expected to affect fertility (Falconer, 2006). Wagner and Oehlmann (2009) have conducted a study to determine the level of endocrine disrupting compounds in usual food stuffs of humans and they selected bottled mineral water as one of the sources of this compounds. The effort was taken based on the fact that endocrine disrupting hormones reaches the body of human mainly through foodstuffs. They used estrogen receptor alpha for the identification. They found that the mineral waters in plastic bottles are seriously contaminated with phthalates that are getting leached into the water from plastic bottle. Thus it was proved beyond doubt that endocrine disrupting compounds are present in plastic wares and extensive use of plastic wares to store food would result in increased level of these compounds in the foodstuffs with a deleterious effect on fertility. According to Rhind (2005) there is an urgent need to study the effects of endocrine disrupting compounds on animals. Very little is known regarding the concentration of these compounds in the different tissues of animals, the concentration required to produce a deleterious effect on the animals, effect of prolonged exposure to an single compound, the effects of different classes of compounds, effect of the exposure to more than one compounds at a time etc. With the available information it is possible to establish that the endocrine disrupting compounds in the environment is affecting human health adversely with a high impact on fertility. The effect of these endocrine disrupting compounds on human reproduction is different for different compounds. Compounds such as diethylstilbestrol affect female reproductive system and cause abnormal follicular growth, ovulation, abnormal formation of corpus luteum and the overall maintenance of ovary would be affected. It would also affect the normal sexual differentiation in females. Pregnancy would be affected because of the negative effects on fertilization and implantation of the embryo in the uterus. Another pollutant called dioxin has been reported to cause endometriosis in women, which is a very painful disease that leads to infertility (Crisp et al. , 1998). There are some compounds, which are naturally occuring such as phytoestrogens produced by plant that could mimic the properties of estrogens produced by humans (Caserta et al. , 2008). Natural sex hormones are used extensively for different purposes in farms as well as in urban areas and there is every chance that these would become harmful to non-targeted organisms including humans because of the concentrating effect. There are many evidences, which prove that environmental contaminants are causing problems in female fecundity as well as fertility (Louis et al. , 2006). There are evidences to prove that puberty, menstruation, endometriosis, pregnancy, senescence period for reproduction etc are affected by exposure to these compounds. Diethyl stilbestrol was given to pregnant women during 1950’s to prevent miscarriage. But later on due to the adverse effects of these compounds most of the kids developed abnormalities. Finally the compound was withdrawn from the market. The female child produced in such cases developed menstrual abnormalities, vaginal hypoplasia, sudden abortion, premature delivery, uterine malformation and overall low fertility. If the child is a male, it was found to develop testicular dysgenesis syndrome (Milhan 1992). Maternal exposure during pregnancy and exposure to these compounds present in the mother’s milk during the prenatal period are believed to be the reasons for such defects. This occurs due to the lipophilic nature of these compounds, which in turn gets stored in the adipose tissues of the mother. This is one of the strongest evidences of the deleterious effect of these compounds on human reproductive system. There are reports that state that human sperm production has decreased in the past 50 years. Although accurate evidence is not there, the reasons for this decrease is attributed to the presence of endocrine disrupting compounds in the environment (Crisp, 1998). Leydig cells tumors are increasingly believed to be caused by this factor. Same is the case of prostrate cancer. Studies conducted in Coke-oven workers have revealed that there has been an increase of mortality among them due to prostrate cancer due to occupational exposure to these compounds. However more research is required to find out the actual cause of this cancer, whether it is due to endocrine disruption by any chemicals in the environment.

Secret Recipe Franchise Analysis

Secret Recipe Franchise Analysis Executive Summary Secret Recipe, Dubai, UAE will be a franchise of catering company called Secret Recipe owned by ALAMERI Group. The business is in food services industry and has successfully established its brand name in Malaysia, Singapore, Indonesia, Thailand, China, Philippines, Pakistan, Brunei, and Australia by virtue of its fine quality cakes, fusion food and distinctive service. The purpose for this business plan is to provide a written guide for managing the franchise of Secret Recipe, Dubai, UAE and seek financing from relevant institution and investors by providing detailed funding business plan. This franchise of secret recipe will be owned by Taleb AlAmeri and will be primarily involved in the provision of variety fresh food products such as cheese, chocolate and fresh, cream cakes, health cakes and brownies. The business mission statement is to be solution oriented innovatively and the vision is to be one of the leading telecommunication companies in Africa. The objective of the company is to provide job opportunities youths, generate income for owners and to give 10% of the revenue back to the community. The fundamentals of this business success rest with the capacity to deliver value to the customer at competitive prices (Ajami, Cool Goddard, 2006).With the background knowledge of stiff competition in the food industry within Dubai, this franchise aims at attracting and retaining its customer through the provision of fine quality food products and distinctive service. In addition to the above, the fundamentals of this business will encapsulate best demonstration of ethical practice within both the internal and external environment. This is because of the backdrop knowledge of the stringent measures and legislations that govern the food industry. The food industry is expected to grow in the near and innovative approaches to food service and delivery will define success within this industry. Changing consumer demographics and increasingly complicated customers are pushing food service businesses to the drawing boards (Toyne Nigh, 1999). The success of this franchise will depend on the capacity to create a powerful market niche that cannot be broken. General Company Description What makes the business very unique is the realization that businesses and consumers are increasingly demanding more leading edge in service and product delivery in the most efficient, time saving and reliable way (Hill, 2008). The Secret Recipe situated at Dubai Mall, UAE a franchise of Secret Recipes will be primarily involved in the provision of fine quality cakes and fusion food. This will encompass the provision of more than 20 types of fusion food, 40 cake creations and pastries, with a flavorful range of ice cream and beverages. Mission statement: To provide world class food services to the customer. Vision: To become a leading provider in fine quality cakes and fusion food. The company will offer value addition services innovatively using the existing technology for reliability and efficiency. With a powerful website to be developed, the customers will be able to sample and purchase their preferred choice of quality fine cakes and fusion foods online. The website will be used to advertise and sell the products and services offered by the company. Company Goals and Objectives Goals To have the franchise operating exactly three months after receiving adequate funding. Obtain $ 200,000 of capital by 31st December 2010 to staff and launch the business towards achieving its long term goals. To attain the stated year goal of recruiting employees by the beginning of the year 2011. Objectives Achieve excellent levels of high ethical standards within the food industry. Have a healthy, successful company that is a leader in customer service and that has a loyal customer following. Achieve a sales target of $20,000 within the first three months of the operation. Business Philosophy: The most important aspect of this business is to provide value to the customer through distinctive service and in a manner that guarantees their return. The more than 20 types of fusion food, 40 cake creations and pastries, with a flavorful range of ice cream and beverages will be sold to all customers of different backgrounds in United Arabs Emirates. However, I envisage to draw the larger bulk of my customer base from the residents of Dubai City. Because of the increasing innovative marketing approaches, the marketing strategy will strive to reach customers of diverse backgrounds and will then be narrowed down to touch on the specific market niche. Food industry is a growth industry that has leaped from one phase to the other and is expected to record tremendous growth in the future. Changes that will arise in the future will mainly be in regard to changing demographics and the complexity in the demands of the customer. This will also include changes in technology that will define the operations and customer service delivery (Singh Delios, 2005). My company will stay in tandem with the changes in the food industry and at pace with technological advancement to reap the most out of the benefits presented by these two growth scenarios. My greatest strength and competency that will also be my competitive advantage is the unrivalled experience in the food industry and chain management. As the owner of this franchise, I plan to put in an aggressive and innovative marketing campaign within this industry that will immediately smoke away competition and enhance my entry and strong regional presence in Dubai. I strongly believe my competitors strength will be based on tears of operation that have made them acquire a deeper sense of understanding of the customer. In fact, after an intensive research on the competitors strengths and weaknesses, the company realized that the competitors strengths are based on the experience, location, advertisement, employee, technology, financial, political connection and goodwill. I plan to plough in my vast experience in management within the food industry to his business and ensure its survival. In addition to the above, my capacity to raise capital that will cover important business start-up areas will provide me a competitive advantage against my competitors who has struggling with waning confidence in small and medium sized entrepreneurs by banks. Furthermore, I expect to reap from the efforts of the main company in regards to brand and image building. This will be a sole proprietorship type of business. This is because I believe the inability to raise adequate capital forms the reason behind enlargement and partnerships. Capital is not a problem in my case. Furthermore, sole proprietorship offers the best and simplest environment in regard to decision making (Cherunilam, 2004). I plan to execute my decisions and discharge my duties within this business without any opposition and inhibition. Products and Services This will be an entirely food business that will only deal with the products of the mother company. I will sell the more than 20 types of fusion food, 40 cake creations and pastries, with a flavorful range of ice cream and beverages offered in all  Secret Recipe  outlets. After an intensive research on the competitors strengths and weaknesses, the company realized that the competitors strengths are based on the experience, location, advertisement, employee, technology, financial, political connection and goodwill. These will form the areas that will define my competitive disadvantages. As a new franchise, I plan to get into the market at a slightly reduced sales fee in comparison to my competitors. This will form the introductory offer of the business that is aimed at drawing the customer to the business. Marketing Plan My marketing ill revolve around the need to adapt my brands to meet local and regional culture because of the understanding that branding act as a means of linking items that are part of product line and emphasizes the individuality of product items. This emphasis can only be achieved in instances where products items fit into the local or regional culture of the target market. This is a food industry and therefore I expect very little conflict with the culture of the people in Dubai. In addition to the above, the adapting of these brands to fit into the local and regional culture of the target market is a competitive advantage that is commonly used by competing firms engaged in the sale of similar or substitute products. In essence, this means that a firm that outperforms the others in the primary goal of performance-profitability-has competitive advantage. A reference back to the branding generic model of firms can be made, where the question of whether firms are branding strategies is cost driven or value added arises. Value added competitive advantage arises in instances where competing firms attach strong cognizance to the understanding of local or regional culture before launching their product lines. Companies succeed in branding strategies that their rival because their products are positioned to capitalize on their unique characteristics of a local or regional group and which, in one or more aspects, their rivals find hard to emulate. This competitive adv antage gives it a basis for outperforming competitors because of the value that firms are able to present to the customer. These will define my marketing plan. Management and Organization As I have stated, the day to day management of the business will be my primary duty with the support of other individuals that will form part of employees in the company. The procedure of running the business will be through delegation as per the chain of current bellow DIRECTORS CEOs SENIOR MANAGER FINANCE MANAGER ASSISTANT AGENTS WATCHMEN HUMAN RESOURSE MANAGER The managers and the staff are very important for the company as they are the means through which goals are realized, duties are carried out and they provide the links between the organization and the clients. 5.2 Management team The initial management team consists of the founders who will be working jointly as shareholders. The founders of the company share a vision for the success of Secret Recipe Franchise situated in Dubai, UAE. Their duty will involve day to day running of the business which involves finance, logistics and human resource. In future the company will have a general manager, finance manager and human resource manager who must have a degree or diploma with the relevant course from a recognized University or College. 5.3 Other personnel Apart from the management team, the company is having three (3) supervisors at the head office and one (1) assistant supervisor in every department. The company is planning to employ watchmen, office messenger and more assistant agents as we will be expanding. These personnel have and will have minimum qualification of O level education and diverse knowledge in the food industry. 5.4 Recruitment The management team has been able to recruit other personnel through networking and train them within the already existing outlets before they are posted to the new outlets. The business will also consider advertising through posters, internal memo, friends and relatives, internet, electronic media like radio and printed media like newspapers. The recruitment process will involve short-listing of the applicants, interviewing of the shortlisted applicants, selection of the successful applicants and finally issuing the appointment letter. We will also recruit through confirmation of the interns and the volunteers. 5.5 Orientation, training and developing of staff Secret Recipe Franchise Company will carry out orientation, training and development of staff through induction, on the job training, contract training, refresher courses, internship and evaluation after training. The company is looking forward to become custodians of a very fragile yet integral good: customer trust, therefore the company will maintain refresher courses to ensure all our staffs are properly aligned when it comes to personal integrity. 5.6 Remuneration/incentives 5.6.1 Salaries/wages The company will offer to the personnel a very competitive basic salary according to the qualification, experience, position and expertise. The salary will also be based on what the competitors offer and the companys returns. There will be a commission for the sales personnel and prompt payment for wages. 5.6.2 Fringe benefits Incentive is also another factor that the company has put at the top of priorities. Incentives will enable, motivate or encourage a particular course of action which in turn will contribute to the company success. We will offer allowances in terms of overtime, leave, millage, hardship and many more as they may arise. There will be bonuses including awards in terms of value for money and certificates. Currently the company provides tea, soft drink and has installed a television system. The business will soon embark on taking our staff to the trips, have insurance cover for them and remit contributions on their behalf to their respective insurance companies. 5.7 Support services The company will require the following support services; financial advisor, legal advisor, banking system, security support services, infrastructure and insurance services. 9. FINANCIAL PLAN The company has at its disposal a sum total of $20,000 as a financial source from directors personal savings. We are therefore looking for funding from other investors and banks. The business finance will be used to for the facilities, equipments, materials, cash for operating expenses and salaries, fees and other costs. 9.1 Capitalization 9.1.1 Own contribution Capitalization based on the shareholders contributions is $20,000. 9.1.2 Proposed funds from borrowing sources For the business to operate as proposed, the start-up cost must be met. The company is therefore proposing funds from borrowing sources of $ 20, 000 9.1.3 Total investment Total investment is the sum of own contribution and the proposed funds from borrowing sources which is $40,000. The following sections lay out the details of the financial plan for rapid, but controlled growth for the next one year. The simple structure to be adopted by the company will provide a great deal of flexibility resulting in few coordination problems hence quick reaction to changes in the market. 7.3 Cash flow projection for the year 2011 Jan Feb Mar April May June July August Sep Oct Nov Dec Total CASH IN Cash carried Forward 300 1,866.67 1,733.34 1,800.01 1,851.68 1,903.35 2,005.02 2,056.69 2,088.36 2,180.03 2,216.7 2,303.37 22,305.22 LOAN 2,000 0 0 0 0 0 0 0 0 0 0 0 2,000 Cash from Sales 200 600 800 850 850 900 900 900 1,000 1,000 1,050 1,100 10,150 CASH OUT Salary 350 450 450 510 510 510 510 510 550 550 550 550 6,000 Rent 40 40 40 45 45 45 45 45 45 50 50 50 540 Office Running Costs 50 50 50 50 50 50 50 70 70 70 70 70 700 LOAN PAYMENT 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 1,120 Drawings 100 100 100 100 100 100 150 150 150 200 200 200 1,650 CASH BALANCE 1,566.67 -133.33 66.67 51.67 51.67 101.67 51.67 31.67 91.67 36.67 86.67 136.67 2,140.04 CUMULATIVE 1,866.67 1,733.34 1,800.01 1,851.68 1,903.35 2,005.02 2,056.69 2,088.36 2,180.03 2,216.7 2,303.37 2,440.04 NOTE: All values are in $ (00) 7.3 Cash flow projection for the year 2011 Jan Feb Mar April May June July August Sep Oct Nov Dec Total CASH IN Cash carried Forward 2,440.04 2,487.79 2,535.54 2,583.29 2,631.04 2,678.79 2,726.54 2,774.29 2,822.04 2,869.79 2,917.54 2,965.29 32,431.98 LOAN 0 0 0 0 0 0 0 0 0 0 0 0 0 Cash from Sales 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 1,099.58 13,195 CASH OUT Salary 650 650 650 650 650 650 650 650 650 650 650 650 7,800 Rent 58.5 58.5 58.5 58.5 58.5 58.5 58.5 58.5 58.5 58.5 58.5 58.5 702 Office Running Costs 58.33 58.33 58.33 58.33 58.33 58.33 58.33 58.33 58.33 58.33 58.33 58.33 700 LOAN PAYMENT 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 93.33 1,120 Drawings 200 200 200 200 200 200 200 200 200 200 200 200 2,400 CASH BALANCE 39.42 39.42 39.42 39.42 39.42 39.42 39.42 39.42 39.42 39.42 39.42 39.42 473.04 CUMULATIVE 2,487.79 2,535.54 2,583.29 2,631.04 2,678.79 2,726.54 2,774.29 2,822.04 2,869.79 2,917.54 2,965.29 3,013.04 NOTE: All values are in $ (00) 7.3 Cash flow projection for the year 2012 Jan Feb Mar April May June July August Sep Oct Nov Dec Total CASH IN Cash carried Forward 3,013.04 3,196.49 3,379.94 3,563.39 3,746.84 3,930.29 4,113.74 4,297.19 4,480.64 4,664.09 4,847.54 5,030.99 48,264.18 LOAN 0 0 0 0 0 0 0 0 0 0 0 0 0 Cash from Sales 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 1,429.5 17,153.5 CASH OUT Salary 845 845 845 845 845 845 845 845 845 845 845 845 10,140 Rent 76.05 76.05 76.05 76.05 76.05 76.05 76.05 76.05 76.05 76.05 76.05 76.05 912.6 Office Running Costs 75 75 75 75 75 75 75 75 75 75 75 75 900 LOAN PAYMENT 0 0 0 0 0 0 0 0 0 0 0 0 0 Drawings 250 250 250 250 250 250 250 250 250 250 250 250 3,000 CASH BALANCE 183.45 183.45 183.45 183.45 183.45 183.45 183.45 183.45 183.45 183.45 183.45 183.45 2,201.4 CUMULATIVE 3,196.49 3,379.94 3,563.39 3,746.84 3,930.29 4,113.74 4,297.19 4,480.64 4,664.09 4,664.09 5,030.99 5,214.44 NOTE: All values are in $ (00) 9.6 break-even levels Cheese Cakes Chocolate and Fresh Cream Cakes Health Cakes Brownies Revenue 6,342,000 1,080,000 1,000,000 1,008,000 720,000 Selling Price (SP)/unit 32 56 32,250 70 80 Units 198,188 19,286 31 14,400 9,000 Variable cost/unit à ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦.. 54 28,375 à ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦ 15 Variable cost à ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦.. 1,041,444 879,625 à ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦Ãƒ ¢Ã¢â€š ¬Ã‚ ¦ 135,000 Contribution 6,342,000 38,556 120,375 1,008,000 585,000 Weighted Average Contribution (WAC) = total contribution/total revenue (Johnson Turner, 2003). = $ 80,939.31/$ 101,500 = 0.8 Break Even-Point (BEP) = Fixed cost/WAC = 90,500 Units 9.7 Measurement of profitability 9.7.1 Operating profit margin Operating profit margin = (operating profit/Revenue) x100 First year: Operating profit margin = (1,400/101,500) x 100 = 1.38% Second year: Operating profit margin = (4,730/131,950) x 100 = 3.58% Third year: Operating profit margin = (22,009/171,530) x 100 = 12.83% 9.7.2 Current Ratio Current Ratio = Current assets/Current liabilities Projected Current Ratio by the end December 31, 2011 = 21,000/11,200 = 1.875 9.7.3 Return On Investment (ROI) Return On Investment (ROI) = Net Profit/Investment Return On Investment (ROI) for the ended December 31, 2010 = 1400/23,000 = 0.06

Coping Mechanisms in Tim OBriens The Things They Carried Essay

During the Vietnam war, soldiers were not exposed to the traditional coping mechanisms of our American society, as illustrated in Tim Obrien's The Things They Carried. These men were forced to discover and invent new ways to deal with the pressures of war, using only their resources while in the Vietnamese jungle. It was not possible for any soldier to carry many items or burdens with them, but if something was a necessity, a way was found to carry it, and coping mechanisms were a necessity to survive the war. Anti-depressants, psychiatrists, massages...there are many different things offered in American society today to help individuals fight the stress of life. People are willing to pay hundreds of dollars for medicine and treatments that promise to give them a better life. They will spend hours of their time at a masseuse or a psychiatrist in constant search for relief from the lives they live. During the Vietnam War, however, soldiers were not exposed to any of these traditional "coping mechanisms". Instead, these men were forced to discover and invent new ways to deal with the pressures of war, using only their resources while in the Vietnamese jungle. It was not possible for any soldier to carry many items or burdens with them, but if something was a necessity, a way was found to carry it, and coping mechanisms were a necessity to survive the war. Each soldier had a personal effect, story, or process that helped him wake up each morning and go to battle once again, and it was these personal necessities that enabled men to return home after the war. Stress was caused by the war itself and the continual conditions of battle, as well as the knowledge and guilt of killing another ... ... could not help themselves, they were not going to be helped. If struggle were encountered, men had personalized ways to reconnect with the real world, and if a tragedy were encountered which affected the entire company, they also found a combined way to cope with this pressure. The priorities of men during the war shifted greatly toward emotional connections to people and events other than the war, and it was these connections that helped them survive and return home. Coping with the stress and burden of war is not an easy task for anyone, yet in The Things they Carried, O'Brien depicts men dealing and coping as much as they can, using only their primeval resources. They learn how to cope with the barest necessities in life, and they learn how to make use of the smallest opportunities to obtain the most relief and joy from every moment in life.

William Morris: Influences of Naturalism in His Life and Works Essay

The main driving element in William Morris’s life has been the nature around him and the houses he lived in. The most prominent influence was the Kelmscott Manor. Therefore, I chose to go with Kelmscott Manor’s layout plan that exhibits the â€Å"inspirational garden â€Å" that led to most of his design decisions, a map that depicts the pockets in the manor and how Morris was inspired by it. In addition to this, an original drawing of the Kelmscott Manor’s exterior that depicts how the manor amalgamated within the garden. To reinforce this, I picked a watercolor of the Kelmscott Manor and a photo that captivates the various perspectives of the garden in the manor. Moving on to his designs that interprets his love for nature I picked up the very first of his wallpaper design of the trellis that has a very naturalistic touch to it with the vines which seemed to be an extension of the â€Å"inspiration garden† on to the paper. Also chose one of the wallpapers he designed during the middle of his lifetime and one of his last designs as well. The underlying concept behind picking those was to outline the consistency in his design concept throughout his life. William Morris was a poet , whose poetry and compositions were also inspired by nature, and to depict his poetry in form of naturalism concept I picked a stance from one of his compositions that talks about forest, flora and fauna which directly ties to his underlying concept. Also the compositions he wrote always had engraved borders which was ... ...an picking the artifacts. Although I did learn about William Morris and his designs in my history classes I learnt about the concept behind his design decisions and in depth analysis of Morris’s evolution as a designer only when I started working on this exhibit. It is imperative that you do research before arriving on any decision in regard to putting up any exhibit. Furthermore after analyzing the different options, it is imperative that you have facts to back up your decisions of the artifacts chosen. Every artifact that you pick for your case should have direct relation to your concept and it should be consistent among all artifacts. Overall a lot of thought should be put into the exhibit and the concept must reflect the ideas distinctly.

The Emperor Jones :: Emperor Jones Essays

The Emperor Jones    In Eugene O'Neil's play, The Emperor Jones, he presents a crucial lesson to mankind: one should not pretend to be someone who he is not. Multiple repercussions may occur to someone who denies their background and race. For example, in The Emperor Jones, the character, Brutus Jones, dissembles as a free white man (Jones was really black and was supposed to be in slavery during that time). Because of Jones' denial, he encounters numerous illusions in the forest of his black heritage, which haunt him until he is finally killed by his natives, under the accusation of an insurgence against his people. O'Neil introduces the theme of denial bluntly. In the opening scene of the play, it is clear to the audience, from a nineteenth century perspective, that Brutus Jones' physical features oppose his personal opinion of his individual status. Jones, a colored man, was expected to be a slave during the eighteen hundreds. Ironically, Jones proudly claims to be a white man and is portrayed as a po werful man in this first scene. After O'Neil presents his theme of denial, he supplies following scenes with the consequences of illusions, displaying his true lineage. One apparition Jones encounters is a gang of Negroes chained, working on the road supervised by a white man. The anticipation of the audience is that Jones will assist the white man with managing the slaves. Instead, Jones is ordered to work; subconsciously, he proceeds to the slave work with his fellow natives. Jones finally realizes his actions and shoots the apparition, which immediately disappears. Jones experiences a similar illusion later of chained blacks, sitting in rows, wailing, awaiting their slavery. Intuitively, Jones joins their rhythm and swaying and his cry rises louder than the others. This illusion leaves on its own and Jones advances through the forest. These two apparitions demonstrate that inside, Jones really understands that he is colored, but he cannot admit it. The next two of Jones' illusion s display that the other people realize that Jones is black which aggravates him even more. First Jones confronts a slave auction. He spectates until he realizes that it is he, who is being auctioned. As a result, Jones loses control and goes wild. Finally, Jones witnesses a religious sacrifice, one similar to his native religious. It is not until Jones realizes that the witch doctor is offering him as a sacrifice, to be eaten by the crocodile, that Jones loses control once again.

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