Challenges of Leadership Essay

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

Inoculation of an Egg

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

A modern audience Essay

But her marriage to will is all a practical arrangement as she is good at her job and Will is a skilled craftsman. As this partnership moves on the pair become more successful, they manage to pay back Mrs Hepworth the loan she gave then to get started and we see a more romantic side to Maggie as she saves a flower from her wedding day bouquet.  Ã¢â‚¬Å"I thought I’d press it in my bible  For keep sake† A factor about Maggie that would appeal to a modern audience is that she is very intelligent. She controls the accounts and organises everything at Hobson’s. She taught Will to read and write, because Will was born into the lower class she was not properly educate and so he was illiterate but Maggie changed that by educating him herself:  Ã¢â‚¬Å"I’ll just set you a short copy, for tonight.†Ã‚  Maggie is honest and fair, when Albert and Freddy are trying to get more money than they agreed out of Mr Hobson, she knows he cannot afford what they are asking and so she gets them down to a more realistic price:  Ã¢â‚¬ËœI know perfectly well what father can afford to pay, and it is not a thousand pounds nor anything like a thousand pounds.’ Maggie is also humble because she is not afraid to ask for help when she needs it. She asks Mrs Hepworth for money as her and will haven’t got any from their previous jobs because Hobson didn’t pay Maggie and Will came from a lower class and had little pay, all to get married and start up a good business  Ã¢â‚¬Å"We’ve paid back Mrs Hepworth what she lent us for our start and made a bit o’ brass on top o’ that.†Ã‚  At the beginning of the play Will lacked self-confidence and he was poor. He was also a victim of Hobson’s exploitation:  Ã¢â‚¬Å"What does she want to praise workmen to his face for?† But Will is a talented craftsman and a skilled worker which is why Mrs Hepworth complimented him and asked that he makes her shoes in the future:  Ã¢â‚¬Å"These are the best-made pair of boots I’ve ever had. Now, you’ll make my boots in future.†Ã‚  This is a great compliment for a working class man because they were not often praised for their work and didn’t often do so well. This is a point about Will that would appeal to a modern audience.  Will, like many other working class in the 1880’s could not read at the start of the play but due to Maggie’s help he learns how to read and write. By starting up his own business shows he is a good business man and he pays off Mrs Hepworth the money back she lent the couple to get the business started. â€Å"We’ve paid off Mrs Hepworth what she lent us for our start.†Ã‚  Will is from a working class background; he is one of Hobsons boot hands and suffers social prejudice from Maggies sisters Alice and Vicky but now is near equal class to them but they still see him form a lower class and treat him with a lack of respect  Ã¢â‚¬Å"Will Mossop do you know who you are talking to?†Ã‚  As the play progresses Wills confidence increase as he now dares to stand up to Hobson, Alice, Vicky and Maggie  Ã¢â‚¬Å"I’ll take you into partnership and give you a half share†. The Audience may find Wills character comical due to his sayings â€Å"Well by gum†Ã‚  And make the viewers laugh. When Maggie tells Will she wants to marry him he is a bit worried at tries to back down from the proposal because he did not love her, but they get their partnership up and running and as the story continues he realises that he can learn to love and give Maggie the respect she needs to create a successful business. From the start to the end of the play Will Mossop changes a lot. He went from working in Hobson’s cellar to owning his own successful business and being married to Maggie. His main change was that he went from poverty to being a member of the working/middle class. By doing this he has made his way up the social ladder, which was very hard to do in the 19th century. Wills struggle could be compared to a dream going from rags to riches. In my opinion this would be the most important factor about Will that would appeal to a modern audience.  Maggie’s determination to make a success of hers and Willie’s marriage and the business that they start would make her popular with a modern audience because she did not really love Will when they were married, but she had to do something to get away from Mr Hobson, her authoritarian father and she also managed to get her sisters married.

Gaming the Console Response

2121 – Writing for MIT Dr. Tim Freeborn January 31, 2012 Gaming the Console – Rhetorical Essay In Linda Bernstein’s informative article â€Å"Gaming the Console† she presents both sides to the ongoing debate about the possible consequences associated with playing video games. Some experts have concluded that gaming leads to negative outcomes such as an increase in violent behavior, or a decrease in academic and social skills. On the other hand, there is a belief that some video games can have positive educational and physical benefits.Bernstein touches on all of these aspects of gaming and leaves the ultimate decision up to the reader to form his or her own opinion on the matter. Throughout the article, there are three images displayed that effectively support the text and help develop the arguments presented. The initial picture that is splashed across the entire first page of the article seems to represent the type of teen who exhibits the potential for increased aggression as a result of playing video games.The photo depicts a male teenager with spiked hair, dark eyes, pierced ear, and stubble on his lip and chin. These characteristics are typically indicative of a more rebellious or aggressive individual. His concentration is evident in his wide-eyed expression and his grimacing face with his tongue in his mouth. He holds the console in a tight grip as shown by the redness of his skin around his thumbs, which indicates his level of intensity. All of these details compliment the notion that video games may be unknowingly impacting areas of the brain associated with aggression.Professor Craig A. Anderson of Iowa State University describes this as follows: â€Å"The effects aren’t huge or immediately noticeable by a game player, but they tend to increase over time† (Bernstein 14). The player may not feel more aggressive after playing a game, but there may be evidence to suggest the effect is still there none the less. The next image in sequence works well in conjunction with the text and seems to support the argument that gaming can have positive benefits if used appropriately. For instance, in the article eighteen-year-old David A. xpresses, â€Å"It’s a fun way of being with your friends when [you are] home relaxing. [It is] competitive and fun, but I only do it when I have extra time† (Bernstein 14). This opinion appears to be shared by the two male and two female teens shown in the picture. They appear to be having fun while playing the game and getting along. Although they have a look of concentration on their faces, the fact that they are all leaning in the same direction could indicate they are playing towards a common goal, and thus encouraging teamwork.It shows boys and girls playing together which illustrates that gaming can have a social and inclusive benefit, and is not simply for socially awkward children. This image can support the notion that if gaming is not performe d at the exclusion of other things such as schoolwork and sports, it can be a fun pastime. The last image Bernstein integrates into her article could be perceived as support for either side of the debate over the impact of video games. The presentation of a boy with his headset on and console in the air with a huge smile indicates again a positive image of gaming.On the other hand, the fact that he is wearing headphones may signify that he is a serious gamer who spends a lot of time gaming at the exclusion of other educational activities. This picture is rather ambiguous because depending on whether he is part of the 8% teen gamers who are addicted or 92% who are not addicted it can have a very different meaning (Bernstein 14). Overall, this image can be interpreted to support whatever argument you are rooting for by the end of the article.Bernstein is successful in presenting useful images that enhance the text while still being fair and showing all views discussed throughout the a rticle. The first image portrays the aggressive nature of gaming, the second illustrates the social aspect of gaming and the third image is left up for interpretation by the viewer. Overall, the article displays a balanced perspective complimented with appropriate corresponding images that support Bernstein’s thesis. Works Cited Bernstein, Linda. â€Å"Gaming the Console. † Current Health Teens. † March 2012.

Renaissance Choir Finance Management System

I will be making a system for the accounts of Renaissance Choir. Renaissance Choir are based in Emsworth and have 29 members. They sing at many events throughout the year and sometimes in Chichester Cathedral. They meet weekly. Statement of Problem Renaissance Choir require a system to manage their finances. It must be flexible and easy to use, so that anyone could come along and use it, without previous knowledge. Schedule of Activities Section Weeks Specification 1.5 Implementation 3 Testing 1.5 Evaluation 1 User Documentation 1 Total 8 Background The current system is based on two books containing input and output for the choir. All entries are written into the book by hand, and the books have to be carried to meetings when necessary and this is not very convenient, and rather slow. A computerised system would be more efficient. The system needs to deal with petty cash, as a constant amount is usually kept as a float or for occasions where cash is required and not a cheque or other method of payment. Overview As the system needs to contain columns of data, a spreadsheet would be most appropriate. I would use Excel, as I am well acquainted with it and it has many useful features that I can use to my advantage. My system would be composed of several sheets- a main sheet with buttons to move between sheets, then other sheets for data entry, listing the entries and making graphs and pivot table reports. Each sheet would have a button to link back to the main menu, and to other relevant sheets. For instance the data entry screen would have a button to submit the data into the spreadsheet, which would activate a macro and move the data. It would also have a button to move to the data-listing screen, which would have a button to link back. The graph page would have a button that would create a graph, and another to clear it. The exit button on the main screen may also clear the graph as well as exiting. Interview and Research Summary I gave a questionnaire to my end user, who filled it in. This has given me much background and other useful information, that can be used to help produce the system. Volume of Data The system will be updated on a weekly basis, so it must be relatively easy to add, delete and change records, as it will be used so frequently. However, cheques need to be paid in at random intervals, and the system will need to recalculate totals and other formulae after each of these events. It must also be able to cope with petty cash, which can fluctuate. Problems with the Current System Problems with the current system are its inability to transfer data easily between the two books and the balance sheet, the fact that it is slow and rather inefficient, due to having two heavy books to carry around. Hardware Full Potential To get good performance from the system, the requirements would be: 200 MHz processor speed 16 MB Ram 16 Bit colour graphics or better Software Full Potential The features of Excel that I will use to create the system are: macros, buttons and controls, formulae, charts, pivot table reports, visual basic, linked workbooks and Input and Output The user will enter data every week. The volume of data entered will depend upon how many events were held in that month, as each event will probably have inputs to the system in income and expenditure. At the end of each month and at the end of the year, the data will be summarised and the summary probably printed. Also, the system will process the lists of data to produce sub-totals and totals over different time spans. User Skill Level My end user is pretty skilled with computers, as she uses them in her work frequently. Therefore the system will not have to explain basic computer functions or other very basic ideas in the user guide, and the technical guide can be pretty complicated. Software Use I will produce the system, but on the main page I will hide the gridlines. I may do this on other pages as well, so that unnecessary gridlines are not shown- if I want gridlines to be shown, then borders can be put around required cells where tables are required. Also, anything that shouldn't be altered (e.g. integral system parts) will be protected to prevent alteration. There will be macros to do things like moving between sheets and adding records. Objectives to Perform My end user wants the system to be able to: * Hold both income and expenditure in one place * Cope with petty cash that is used for floats at events and other purposes * Be able to transfer data between sections easily * Produce graphs and charts of the data * Produce summaries of the data Performance Indicators * Must be able to open relatively quickly, even on lower spec machines * Must be able to contain a large volume of data while still retaining a file size below 100 kilobytes * It must be relatively easy and quick to enter data or perform other operations * It must be relatively difficult to make mistakes while entering data (using drop-down lists, buttons, macros etc) * It must be easy to get to specific sections * It must be foolproof- e.g. impossible to alter critical system aspects * It must be possible to alter the structure of the database when necessary

Affirmative action plan Assignment Example | Topics and Well Written Essays - 3000 words

Affirmative action plan - Assignment Example g that equal rights and treatment are present within the workplace1.The purpose of affirmative action plans is to ensure that there is equal opportunity for all people. However, this approach differs from a traditional equal opportunity approach, because equal opportunity attempts to eliminate discrimination when it occurs, but affirmative action plans are more active, and aim to prevent discrimination happening2. This is an important distinction and is critical in the way that organizations develop and enact affirmative action plans. Despite their prevalence throughout the United States, and the legislative support that is present for them, there is considerable controversy about the use of affirmative action plans. Opponents argue that they promote discrimination through the process of reverse discrimination, where people who are not part of a minority group have less opportunities and benefits than those who are in a minority. The purpose of this paper is to examine this history surrounding affirmative action plans, their use in present day organizations, the controversy about them and to consider what this is likely to mean for affirmative action plans in the future. People of racial or ethnic minority groups as well as women have often experienced legal or social exclusion in many areas. In the 1960s and 1970s, Hispanic Americans and African Americans were mostly segregated into low income jobs, leaving some categories of employment for white males only, including fire departments and police departments in many major cities. In contrast, roles that required hard manual labor tended to be almost exclusively occupied by people from ethnic minorities. Although there were significant improvements to civil rights, including the Civil Rights Act of 1964, the creation of equal opportunities for women and minority groups was often difficult. This was in part due to the difficulties in implementing the changes and the fact that organizations could easily ignore the

A Structured Stakeholder Analysis Essay Example | Topics and Well Written Essays - 1000 words

A Structured Stakeholder Analysis - Essay Example This paper will review The Richard and Hinda Rosenthal organization as a key stakeholder in healthcare, with regards to how it acts towards solving the issue of microbial resistance. The association of the Rosenthal Family foundation to health care is derived from its connection with the Institute of Medicine, IOM which in 1988 initiated an outreach program. The program aimed at creating a communal innovative outreach through which organizations and other able parties could team up with the IOM and brainstorm together in solving specific issues in health care. The Rosenthal Family Foundation, which at that time was known as the Richard and Hinda Rosenthal Foundation, strongly moved in to support the move by the IOM. Ever since, the two have been working as partners year after year, and have been including additional experts in the fields in addressing some health policy issues. The main operational base of this partnership has been around Washington DC, although their services in health care have at times extended to other parts of the United States (Institute of Medicine, 2014). One of the most recent campaigns hosted by the partnership between the Institute of Medicine and the Rosenthal Family Foundation is titled â€Å"The 2014 Richard and Rosenthal Symposium 2014: Antimicrobial Resistance: A Problem without Borders†. In this campaign, the foundation reveals that it was inspired by a report released by the Centers for Disease Control and Prevention. The report stated that of the five threats currently facing the United States, antimicrobial resistance dominates as one of the most critical. The issue lies in that about two million Americans acquire bacterial infections, which prove resistant to one or more antibiotics. The result of this is approximately twenty-three thousand annual deaths (CDCP, 2014). The Rosenthal Foundation is representing the World Health Organization, which through the White House in 2011