Free Essays on Proctor & Gamble Case Study

Background Proctor & Gamble (P&G) is one of the world’s largest manufacturers of a wide array of products, including many household grocery items – antiperspirants, baby care, household cleaners, skin care, fabric care, food & beverages, laundry, etc. At the end of 2001, P&G had net sales of over $39.2 billion1. P&G is known as a dominant, aggressive developer and marketer of high-quality consumer goods. Their stock prices have returned almost 500% return in the last decade alone.2 They have been in business for over 150 years. Early on, the P&G growth strategy was isolated in three main areas – acquisitions, developing new product brands (as well as further developing existing brands), and globalization. Because of a law-suit in 1957 regarding acquisitions, P&G was forced to focus on new branding and international expansion over the next twenty years3. Focusing on the international aspect of business, P&G aggressively expanded their domestic household products by merely duplicating the U.S. products and marketing them into global markets. From 1953 to 1985, P&G went from an International portfolio of zero to $4 billion. Ed Artzt, the president of P&G International, refined their products by actually tailoring them to each individual global market. This refinement of marketing enabled P&G to jump to $15 billion in International sales by 1993. Ed Artzt became the CEO of P&G in 19904. Regarding the development of existing and new brands, P Food/Beverage; Paper; Soap; and Special Products (i.e. chemicals). Within each sector were product categories. Each category was then in charge of a group of brands. Interestingly, in the 1980’s P&G, as well as other manufacturers, grew to accept the ‘forward buying’ of promoted merchandise. By the mid 1980’s, the industry stocked a 3 month supply of many brands5. This was obviously a tremendous disadvantag... Free Essays on Proctor & Gamble Case Study Free Essays on Proctor & Gamble Case Study Background Proctor & Gamble (P&G) is one of the world’s largest manufacturers of a wide array of products, including many household grocery items – antiperspirants, baby care, household cleaners, skin care, fabric care, food & beverages, laundry, etc. At the end of 2001, P&G had net sales of over $39.2 billion1. P&G is known as a dominant, aggressive developer and marketer of high-quality consumer goods. Their stock prices have returned almost 500% return in the last decade alone.2 They have been in business for over 150 years. Early on, the P&G growth strategy was isolated in three main areas – acquisitions, developing new product brands (as well as further developing existing brands), and globalization. Because of a law-suit in 1957 regarding acquisitions, P&G was forced to focus on new branding and international expansion over the next twenty years3. Focusing on the international aspect of business, P&G aggressively expanded their domestic household products by merely duplicating the U.S. products and marketing them into global markets. From 1953 to 1985, P&G went from an International portfolio of zero to $4 billion. Ed Artzt, the president of P&G International, refined their products by actually tailoring them to each individual global market. This refinement of marketing enabled P&G to jump to $15 billion in International sales by 1993. Ed Artzt became the CEO of P&G in 19904. Regarding the development of existing and new brands, P Food/Beverage; Paper; Soap; and Special Products (i.e. chemicals). Within each sector were product categories. Each category was then in charge of a group of brands. Interestingly, in the 1980’s P&G, as well as other manufacturers, grew to accept the ‘forward buying’ of promoted merchandise. By the mid 1980’s, the industry stocked a 3 month supply of many brands5. This was obviously a tremendous disadvantag...

Calculating Number of Days - Mathematics

Calculating Number of Days - Mathematics An interest period will involve two dates. The date the loan is given and the end date. You will need to find out from the loan institution if they count the day the loan is due or the day before. This can vary. In order to determine the exact number of days, you will first need to know the number of days in each month. January - 31February - 28*March - 31April - 30May - 31June - 30July - 31August - 31September - 30October - 31November - 30December 31 You can remember the number of days in a month by memorizing the days of the months nursery rhyme: Thirty days hath September,April, June, and November,All the rest have thirty-one,Excepting February alone,Which has but twenty-eight days clearAnd twenty-nine in each leap year. February and Leap Year We cannot forget about Leap Year and the changes it will present for the number of days in February. Leap years are divisible by 4 which is why 2004 was a leap year. The next leap year is in 2008. An extra day is added to February when February falls on a leap year. Leap years also cannot fall on a centennial year unless the number is divisible by 400 which is why the year 2000 was a leap year. Lets try an example: Find the number of days between Dec. 30 and July 1 (not a leap year). December 2 days (Dec. 30 and 31), January 31, February 28, March 31, April 30, May 31, June 30 and July 1 we dont count. This gives us a total of 183 days. Which Day of the Year Was It? You can also find out the exact day that a specific date falls on. Lets say you wanted to know what day of the week a man walked on the moon for the first time. You know that it was July 20, 1969, but you dont know which day of the week it falls on. Follow these steps to determine the day: Calculate the number of days in the year from Jan. 1 to July 20 based on the number of days per month above. You will come up with 201 days. Subtract 1 from the year (1969 - 1 1968) then divide by 4 (omit the remainder). You will come up with 492. Now, add 1969 (original year), 201 (days prior to the event -July 20, 1969) and 492 to come ups with the sum of 2662. Now, subtract 2: 2662 - 2 2660. Now, divide 2660 by 7 to determine the day of the week, the remainder the day. Sunday 0, Monday 1, Tuesday 2, Wednesday 3, Thursday 4, Friday 5, Saturday 6. 2660 divided by 7 380 with a remainder of 0 therefore July 20, 1969 was a Sunday. Using this method you can find out which day of the week you were born on! Edited by Anne Marie Helmenstine, Ph.D.

Impact of Financial Losses for GM Auto Manufacturers Essay

Impact of Financial Losses for GM Auto Manufacturers - Essay Example By mid-century, the automobile was a necessity in every U. S. household, and GM led the way with Ford and Chrysler close behind. The Big Three controlled the industry from Detroit, Michigan. According to Wikipedia, the period from 1960-1985 was perhaps the "greatest in GM's history, as it eventually held slightly over 50% of the U.S. Market" (General Motors, 2006, 2.3). At the time, it was all about status, having the most popular brand. Unfortunately, in the mid 1990s, a downward spiral began, which has yet to be resolved for American car makers. It could have been predicted in 1984 when a joint venture between GM and Toyota gave Toyota an opportunity to establish a base in the United States and avoid newly established tariff on foreign pick-up trucks. Toyota's growth has accelerated ever since, with a $4.1 billion dollar gain in 2005 compared with GM's $10.6 billion dollar loss ( Solman, 2006). In a recent PBS interview, GM CEO Rich Wagoner noted that restructuring is taking place in the company in an effort to "compete in the global auto industry and global economy" (Solman, 2006, par. 12). He claims that the company is launching new products, and accelerating the application of biofuels E85. Wagoner also mentions a "breakthrough" health care deal with United Auto Workers (UAW) and the major restructuring of GMAC. With attrition and plant closings, he is optimistic that the company will be more streamlined and better able to compete globally. What sounds like positive action, however, definitely has its down side, with employees about to be faced with increased co-pay for health care and pharmacy and heavy job losses as plants close down. The company's "failure to foresee drop in demand for gas-guzzling SUVs, slow entry into hybrid market and Toyota's reputation for high quality" puts them behind in the race for market share (Kellar, 2006, par. 2). Micheline Maynard, Detroit bureau chief for The New York Times, claims in her book, The End of Detroit (2003) that by focusing on high-profit light trucks, American automakers, including General Motors, turned its back on people wanting to own cars rather than trucks and opened the door to Toyota, Honda and Hyundai. Consumers retaliated by turning their backs on trucks and purchasing foreign-brand cars. General Motors obviously does not look beyond present trends to what the future might bring. Krolicki's Reuters article (2006) is only one of several news stories (Wall Street Journal, Bloomberg, CNW) reporting the bankruptcy of Delphi, one of GM's most important suppliers, which, along with GMAC filing errors, brought about even more losses in 2005 than had been previously noted. In keeping with General Motors' short-term goals to increase market share, Associated Press writer Dee-Ann Durbin recently announced the company's gas-price

THE SUBJECT IS SOCIAL WORK Psychsocial history of a charector from a Essay

THE SUBJECT IS SOCIAL WORK Psychsocial history of a charector from a novel, She's Come Undone by Wally Lamb - Essay Example The character of Dolores Price is unlike any other because she drowns herself in her world of fantasy only to surface from time to time into a world of unrealistic problems. In the words of Dolores all these troubles â€Å"began somehow, in 1956 on the day our first television was delivered.† (Wally Lamb, 1992)She had no idea that something as simple as a television would lead her on the path of a personal tumultuous odyssey. The character of Dolores as presented by the author Wally Lamb, was vulnerable yet mysterious human being. To a great extent her family was responsible for the outcomes in her life. Her mother who was an emotionally disturbed person was quite fragile and battled with mental illness and therefore could not be of much help to Dolores. Her father was a lustful man who always threatened to leave the family. Her proud grandmother had a lot of pent up feelings and suffered deeply as she could not express them. Another person who did not belong to her family but was quite responsible for Dolores’ condition, was her handsome neighbor Jack Speight whose final betrayal throws the life of Dolores almost permanently off track. In this novel there are many cultural and social factors that play havoc in her life and brings her to a point of breaking. Quite frequently in the story we find Dolores making encounters with people who mirror the members of her family by whom she faced disappointment throughout her life. Beginning with her father, her path seemed to be guided towards dysfunctional relationships with men. In her quest to come to terms with her own identity, Dolores is victimized by her own circumstances and stands out as a character that is loathed as well as adored because of the unconventional way she presents herself. Dolores the protagonist is a tragic character who encounters tragedy after tragedy all through her life from the

The love for American football Essay Example for Free

The love for American football Essay Someone who is from another country like me, may believe that life in the United States is very diverse from life anywhere else. Countless people from other countries have a completely distorted image of how we may live there in America and the mentalities thatthey all posess. Not all of Americans are spoiled, overweight, or are obsessed with football. All of them are completely different for the most part, and are actually concerned about what is occurring in the world today. However, I do believe I can say that most Americans are proud to be here, and proud to represent the UNITED States, and that pride and love is shared by everyone, kind of like a big family. Just like other countries, there are things that the people tend to be attracted to more than another group of people would. For example, in Mexico, soccer is huge, and almost everyone there is crazy about it, but here in the United States, soccer doesn’t hold near as many fans. Since one doesn’t really know a lot about the life in the United States, and the things all Americans seem to enjoy, I would like to enlighten you to the one thing that I believe is totally ‘American’. And of coarse, I would have to say its football. Now, no all of Americans enjoy the hazardous sport, but about nine out of ten of them absolutely adore it(including myseld). The competition and the thrill that you get from watching breathtaking game, will most likely have you sitting at the edge of your seat, not wanting to miss a minute of whar you are seeing take place on the television screen. The runs, the cheers, the interceptions, the touchdowns, and most of all the victory when it’s all over is what I believe makes football what is it today in the society of Americans. If you were to meet another Amrican, I can almost guarantee that the person has a favorite football team that they support with all of theit hearts.

Academic Effects of Active Parental Involvement Essay -- A Level Essays

Academic Effects of Active Parental Involvement Findings demonstrate that parent involvement in a child’s learning is positively related to their achievement. The first teachers of our children are the adults in the home. This is where the children learn their attitudes and values that are engraved in them for the remainder of their lives. When families as a whole participate, in children’s education in positive ways, there are noticeable changes in the child’s test scores, attendance records, quality of work, attitudes and behavior, graduation rates, and the amount that enroll in higher education. The level of involvement is often questioned by the parents; however, research suggests that â€Å"the more intensively parents are involved in their children’s learning, the more beneficial are the achievement effects† (Cotton and Wikelund, 2002, p. 2). It is believed that when parents monitor homework, encourage participation in extracurricular activities, are active in parent-teacher associa tions, and help children develop plans for their future, children are more likely to respond and do well in school. There are indications that prove that the most effective form of parental involvement is when the parents work directly with their child. Researchers find that the more activity each parent puts in, there will be greater achievement for that student in comparison to the children with passive parents. When parents actively participate, that is work with their children, attend their activities, and help in the classroom, greater achievement is noted. Academic Effects of Passive Parental Involvement Passive parent involvement is better than no involvement, however the effects aren’t as successful for the child. This... ... This article helps to explain the social effects that parental participation has on children. It describes how self-esteem and motivation is related to the amount parental interest in the child and their education. Watkins, T. (1997). Teachers communications, child achievements, and parent traits in parent involvement models. Journal of Educational Research, 91 (Sept./Oct.), 14. Retrieved March 12, 2002. This article explains how parental involvement through home instruction, volunteering in the classroom, has been linked to the quality of children’s learning and motivation. Swick, K. (1984). Inviting Parents into the Young Child’s World. Stipes Publishing Company. Playing Their Parts: What Parents and Teachers Really Mean by Parental Involvement. (1999). Encyclopedia Britannica Online. Retrieved on April 15, 2002 from the World Wide Web.

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

Air pollution in Kawempe Division of Kampala, Uganda

Air pollution in Kawempe Division of Kampala, Uganda Hazard Word pictureIntroductionThe wellness effects of air pollution have been widely documented and assessed largely in North America and Europe, nevertheless air pollution in low- and middle-income states poses a great public wellness load that has non been decently assessed ( 1–6 ) . All right particulate affair comprised of atoms of width 2.5?g or less ( PM2.5) has been associated with mortality due to the development of cardiovascular or respiratory diseases and lung malignant neoplastic disease ( 3 ) . In the Kawempe Division in Kampala ( Uganda ) measurings of PM2.5from December 2013 and January 2014 were found to be above guideline bounds of the World Health Organization ( WHO ) bespeaking the demand for a consistent exposure appraisal of the air pollution and its wellness load in Uganda ( 7,8 ) .Hazard IdentificationAcute events of smog in the Meuse vale in Belgium and in London in 1933 and 1952, severally were among the first events that indicated the association between air pollution and mortality ( 9,10 ) . More late epidemiologic surveies quantified effects of chronic and ague exposures to poisons such as particulate affair, ozone, or benzine on wellness ( 3,11,12 ) . More specifically PM2.5exposure have been associated with inauspicious wellness results in big epidemiologic surveies in assorted scenes ( 8,13 ) . Chronic effects of exposure to particulate affair have been studied in prospective cohort surveies with big sample sizes and long follow-up periods in assorted locations such as the US, Canada, in Europe and China ( 2,4,5 ) . The first survey that associated cardiorespiratory and lung malignant neoplastic disease mortality with PM2.5was published in 1993 by Dockery et Al ( 3 ) . In this first appraisal informations from six metropoliss with changing degrees of air pollution and mortality from cardiorespiratory diseases, lung malignant neoplastic disease, all causes of mortality and mortality from all other causes except cardiorespiratory diseases or lung malignant neoplastic disease was investigated. Following up to the â€Å"six metropoliss study† Laden et Al. in 2006 reported on the association between cardiovascular diseases ( CVD ) mortality, lung malignant neoplastic disease and respiratory upsets ( 14 ) . Following up the same cohort and reanalyzing the information Lepeu le et Al. in 2012 besides confirmed the association between CVD and lung malignant neoplastic disease mortality with PM2.5exposures ( 15 ) . In 2002, Pope et Al. besides reported the consequences of a larger cohort with PM2.5measurings of 51 metropolitan countries in the US, reasoning besides that there is increased hazard of mortality at 4 % , 6 % and 8 % from all causes, CVD, and lung malignant neoplastic disease, severally with each 10?g/m3addition in PM2.5( 4 ) . Similar consequences were besides reported in the Canadian survey of Crouse et al. , 2012 in a cohort of 2.1 million Canadian grownups ( 16 ) . All these surveies set the scene for comprehensive wellness hazard appraisal of PM2.5. They provided cohesive and timely consequences from dependable beginnings on the fluctuations PM2.5and mortality fulfilling Hill’s standards.Exposure AppraisalKampala, the capital of Uganda has a population of about 1,500,000, it is the 2neodymiummost thickly settled territory in the state ( 17 ) . The Kawempe division of Kampala is located in the North of the metropolis and has a population of 338,312, with an estimated 84,240 above the age of 30 old ages ( 17 ) . Measurement of ambient PM2.5degrees took topographic point two yearss during winter 2012-2013 in the country of Mperewre of the Kawempe Division and they were reported as portion on a pilot survey published in 2014 ( 7 ) . The mean Autopsy2.5concentration for the topographic point measurings was 104.3?g/m3. It besides exceeds both the one-year and 24-hour WHO guideline values that are set at 10?g/m3and 25?g/m3, severally, and antecedently reported measurings in developed states ( 8 ) . The mensural PM2.5degrees are nevertheless consistent with measurings in other developing states. Datas from Asiatic metropoliss suggest that the high Autopsy2.5concentrations are non rare in developing scenes where monitoring is non routinely conducted. Harmonizing to the 2010 study of the Health Effects Institute on outdoor air pollution in the underdeveloped states of Asia the degrees of PM2.5can be every bit high as 150?g/m3( 6 ) . In the same study, for illustration, in Shenzhen and Guengzhou, in China, 24-hour mean summer concentrations of PM2.5were 35?g/m3Autopsy2.5was every bit high as 97.5?g/m3. Exposure appraisal of PM2.5effects on mortality in the Kawempe Division are hindered by the deficiency of one-year norm values that would reflect fluctuations in air pollution. However given the features of the country, where a landfill is located and the tendency for increased urbanisation in Uganda and its deductions ( i.e. increased traffic etc ) the high Autopsy2.5degrees in Mperewre may reflect the norm of PM2.5degrees in the location ( 18,19 ) . Within the range of the present exposure appraisal we are based on the premise that the mean of the available topographic point measurings reflects the average PM2.5degrees in the Kawempe division of Kampala. Based on the handiness of information we can non be certain about the PM2.5concentrations fluctuate in the country over clip and the derived decision might be over- or undervaluing the existent wellness effects of PM2.5wellness effects. Table 1 Autopsy2.5concentrations and meteoric parametric quantities on the yearss that measurement took topographic point harmonizing to Schwander et al. , 2014.December 31, 2012January 2, 2013Autopsy2.5 ­concentrations (?g/m ­3 ­)104.9103.7Average concentration (?g/m ­3 ­)104.3Meteorologic parametric quantitiesAverage temperature (0C ) [ min, soap ] 22.2 [ 18.9, 25.0 ]24.4 [ 20.6, 27.8 ]Dew point temperature (0C )18.920.0Mean wind velocity ( kilometers per hour ) [ max ] 8.1 [ 16.1 ]8.1 [ 19.3 ]Exposure-Response AnalysisThe exposure-response relationships between PM2.5and mortality from CVD and lung malignant neoplastic disease have been antecedently studied in US populations by Laden et Al. ( six metropoliss study, follow-up ) and by Pope et Al. ( 4,14,22 ) . In the survey of Laden CVD and lung malignant neoplastic disease deceases were associated with exposure to PM2.5( 14 ) . Specifically a 10?g/m3addition in the mean Autopsy2.5was associated with comparative hazard ( RR ) of 1.28 ( 1.13-1.44, 95 % CI ) for CDV mortality and a RR of 1.27 ( 0.96-1.69, 95 % CI ) for lung malignant neoplastic disease mortality. In the survey of Pope et al. , 2002 for the same PM2.5incremental alteration addition in lung malignant neoplastic disease mortality was estimated at 8 % ( 4 ) . Both surveies, the 1993 survey of the six metropoliss and the Lepeule’s follow up have established the association between increased exposure to PM2.5and increased hazard for C VD and lung malignant neoplastic disease mortality. The form of the exposure-response map nevertheless is non additive in all doses nor does it hold the same form for both results. The exposure-response map of mortality from CVD starts steep in low exposures and going flatter with increasing concentrations ( 22 ) . In the instance of lung malignant neoplastic disease mortality the exposure-response map is more additive and it is, as expected, more influenced by confounders ( i.e. smoke ) ( 22 ) . Additionally in all the aforesaid surveies the exposure-response maps have been established based on the effects of low exposures, normally below 50?g/m3. In Uganda, the age-adjusted mortality rate from lung malignant neoplastic disease is 2.7 among males and 2.4 among females ( per 100,000 ) while the same index for CVD is 276.7 and 250.7 per 100,000 population ( males and females, severally ) ( 20,21 ) . In order to gauge the figure of CVD and lung malignant neoplastic disease deceases attributed to 104?g/m3of ( assumed ) one-year exposure we will be based on the exposure-response maps for CVD and lung malignant neoplastic disease mortality developed by Pope et Al, 2011 that were derived from a prospective cohort of 1.2 million grownups ( & gt ; 30 old ages old ) and old surveies in the US which accounted for assorted confounders of exposure. They estimated the RR associated with different degrees of PM2.5exposure accounting for ambient air pollution, active coffin nail smoke and secondhand smoke to gauge the day-to-day norm inhaled dosage of PM2.5. The RR for each of the two results ( CVD and lung malignant neoplastic disease mortality ) was so calculated with the power map: RR=1+? ( dosage )?which corresponds to a nonlinear monotone map for which at zero dose the RR is equal to 1. Table 2 Exposure appraisal informationPopulation features( 17 )MalesFemalesEntireKawempe Division159,800178,512338,312Population above 30 old ages old ( 24.9 % * )39,79044,44984,240Kampala722,638793,5721,516,210Entire population16,935,45617,921,35734,856,813Age standardized decease rates per 100000( 20,21 )Cardiovascular diseases276.7250.7Lung malignant neoplastic disease2.72.4Entire figure of deceases in Kawempe territory for the those & gt ; 30 old ages oldCardiovascular diseases110111222Lung malignant neoplastic disease112RR estimations for both genders ( for dosage of PM2.5104?g/m3)Attributable hazard fractionNumber of instances attributed to PM2.5Cardiovascular diseasesRR=1+0.2685 ( dosage )0.27301.950.49108Lung malignant neoplastic diseaseRR=1+0.3195 ( dosage )0.743311.090.912Hazard Word pictureWith an estimated RR of 1.95 for CVD mortality due to PM2.5degrees of 104?g/m3the PM2.5-associated mortality instances in the entire population above 30 old ages old of Kawempe Division is about 108 ( Table 2 ) . The age standardized mortality rate for CVD – the primary wellness result that has been associated with increased air pollution – in Uganda is comparatively high contrary and future research is needed to decently gauge the fraction of instances attributed to air pollution which was comparatively high in this study ( 0.49, ( Table 2 ) . As expected given the to the low mortality rate of lung malignant neoplastic disease in the country and the fact it is confounded by other factors that could non be assessed in ( i.e. smoking ) we can non properly measure the impact of PM2.5on the figure of lung malignant neoplastic disease deceases in Kawempe Division. For the present analysis the informations used were compiled from assorted beginnings. Population estimations were taken from the probationary study on the 2014 nose count. Lung malignant neoplastic disease and CVD age standardized mortality rates were taken from GLOBOCAN and the WHO, severally. Therefore all computations are based on the premise that for the 2014 of Kawempe Division the mortality rates are the same as the last reported in the aforesaid beginnings. The fact that there is the information might non reflect the decently the features of the population is an of import restriction of this appraisal. Along with the fact that hazard estimations were based on surveies of lower Autopsy2.5degrees. Future research needs to be based on seasonably collected information and hazard theoretical accounts that account for the high exposures. Mentions 1. Katsouyanni K, Rimm EB, Gnardellis C, Trichopoulos D, Polychronopoulos E, Trichopoulou A. Reproducibility and comparative cogency of an extended semi-quantitative nutrient frequence questionnaire utilizing dietetic records and biochemical markers among Grecian school teachers. Int J Epidemiol. 1997 Jan 1 ; 26 ( suppl 1 ) : S118. 2. Raaschou-Nielsen O, Andersen ZJ, Beelen R, Samoli E, Stafoggia M, Weinmayr G, et Al. Air pollution and lung malignant neoplastic disease incidence in 17 European cohorts: prospective analyses from the European Study of Cohorts for Air Pollution Effects ( ESCAPE ) . The Lancet Oncology. 2013 Aug ; 14 ( 9 ) :813–22. 3. Dockery DW, Pope CA, Xu X, Spengler JD, Ware JH, Fay ME, et Al. An Association between Air Pollution and Mortality in Six U.S. Cities. New England Journal of Medicine. 1993 Dec 9 ; 329 ( 24 ) :1753–9. 4. Pope III CA. Lung Cancer, Cardiopulmonary Mortality, and Long-run Exposure to Fine Particulate Air Pollution. JAMA. 2002 Mar 6 ; 287 ( 9 ) :1132. 5. Cao J, Yang C, Li J, Chen R, Chen B, Gu D, et Al. Association between long-run exposure to outdoor air pollution and mortality in China: A cohort survey. Journal of Hazardous Materials. 2011 Feb 28 ; 186 ( 2–3 ) :1594–600. 6. HEI International Scientific Oversight Committee. Outdoor Air Pollution and Health in the Developing Countries of Asia: A Comprehensive Review. Boston, MA: Health Effects Institute ; 2010. 7. Schwander S, Okello CD, Freers J, Chow JC, Watson JG, Corry M, et Al. Ambient Particulate Matter Air Pollution in Mpererwe District, Kampala, Uganda: A Pilot Study. J Environ Public Health [ Internet ] . 2014 [ cited 2015 Feb 26 ] ; 2014. Available from: hypertext transfer protocol: //www.ncbi.nlm.nih.gov/pmc/articles/PMC3945229/ 8. WHO | Air quality guidelines – planetary update 2005 [ Internet ] . WHO. 2015 [ cited 2015 Mar 5 ] . Available from: hypertext transfer protocol: //www.who.int/phe/health_topics/outdoorair/outdoorair_aqg/en/ 9. Bell ML, Davis DL. Reappraisal of the deadly London fog of 1952: fresh indexs of ague and chronic effects of acute exposure to air pollution. Environ Health Perspect. 2001 Jun ; 109 ( Suppl 3 ) :389–94. 10. Nemery B, Hoet PH, Nemmar A. The Meuse Valley fog of 1930: an air pollution catastrophe. The Lancet. 2001 Mar ; 357 ( 9257 ) :704–8. 11. Kheirbek I, Wheeler K, Walters S, Kass D, Matte T. PM2.5 and ozone wellness impacts and disparities in New York City: sensitiveness to spacial and temporal declaration. Air Qual Atmos Health. 2012 Oct 12 ; 6 ( 2 ) :473–86. 12. Savitz DA, Andrews KW. Review of epidemiologic grounds on benzine and lymphatic and haematopoietic malignant neoplastic diseases. Am J Ind Med. 1997 Mar 1 ; 31 ( 3 ) :287–95. 13. US EPA O. Particulate Matter | Air & A ; Radiation | US EPA [ Internet ] . 2013 [ cited 2015 Mar 9 ] . Available from: hypertext transfer protocol: //www.epa.gov/pm/ 14. Laden F, Schwartz J, Speizer FE, Dockery DW. Decrease in Fine Particulate Air Pollution and Mortality. Am J Respir Crit Care Med. 2006 Mar 15 ; 173 ( 6 ) :667–72. 15. Lepeule J, Laden F, Dockery D, Schwartz J. Chronic Exposure to Fine Particles and Mortality: An Drawn-out Follow-up of the Harvard Six Cities Study from 1974 to 2009. Environmental Health Perspectives. 2012 Mar 28 ; 120 ( 7 ) :965–70. 16. Crouse DL, Peters PA, new wave Donkelaar A, Goldberg MS, Villeneuve PJ, Brion O, et Al. Hazard of Nonaccidental and Cardiovascular Mortality in Relation to Long-term Exposure to Low Concentrations of Fine Particulate Matter: A Canadian National-Level Cohort Study. Environmental Health Perspectives. 2012 Feb 7 ; 120 ( 5 ) :708–14. 17. UBOS. National Population and Housing Census 2014 – Probationary consequences [ Internet ] . Uganda Bureau of Statistics ; 2014 Nov. Available from: hypertext transfer protocol: //www.ubos.org/onlinefiles/uploads/ubos/NPHC/NPHC 2014 PROVISIONAL RESULTS REPORT.pdf 18. Mwiganga M, Kansiime F. The impact of Mpererwe landfill in Kampala – Uganda, on the environing environment. [ Internet ] . Makerere University – College of Agricultural and Environmental Sciences. 2012 [ cited 2015 Mar 9 ] . Available from: hypertext transfer protocol: //caes.mak.ac.ug/research/research-publications/141-publications-for-2005/820-the-impact-of-mpererwe-landfill-in-kampala-uganda-on-the-surrounding-environment.html 19. Mwiganga M, Kansiime F. The impact of Mpererwe landfill in Kampala–Uganda, on the environing environment. Physicss and Chemistry of the Earth, Parts A/B/C. 2005 ; 30 ( 11–16 ) :744–50. 20. WHO. Noncommunicable Diseases ( NCD ) Country Profiles – Uganda [ Internet ] . 2014 [ cited 2015 Mar 9 ] . Available from: hypertext transfer protocol: //www.who.int/nmh/countries/uga_en.pdf? ua=1 21. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et Al. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [ Internet ] . GLOBOCAN 2012 v1.0. 2013 [ cited 2015 Mar 5 ] . Available from: hypertext transfer protocol: //www.who.int/phe/health_topics/outdoorair/outdoorair_aqg/en/ 22. Pope CA, Burnett RT, Turner MC, Cohen A, Krewski D, Jerrett M, et Al. Lung Cancer and Cardiovascular Disease Mortality Associated with Ambient Air Pollution and Cigarette Smoke: Shape of the Exposure–Response Relationships. Environmental Health Perspectives. 2011 Jul 19 ; 119 ( 11 ) :1616–21. Appendix – Analytica theoretical account File: Xanthi_Andrianou_RA_W4_Risk_characterization

Little Prince Reaction Paper Essays

Little Prince Reaction Paper Essays Little Prince Reaction Paper Essay Little Prince Reaction Paper Essay Essay Topic: The Prince It is only with the heart that one can see rightly. What is essential is invisible to the eye. This line struck me the most in this book because it is definitely real. It is important to see things not only through the eyes but through the heart as well. Seeing through the heart is like looking at life with love. Life has many problems and by seeing things differently, it would make everything feel better. Looking at things in a different perspective creates a big difference. People usually tend to take for granted those people who love them.They usually look for others whom they do not really love, but whom they want. I learned a lot of things from this book because it is all about the adventure of a little prince who went to different planets to forget about his rose and the people he met in his journey, including the narrator. He learns the secret of the importance of life on planet Earth. The book also emphasizes the difference between the minds of the children and adult. The adult tend to see things in a different way that coincides with the point of view of the children.In the case of the narrator, his childhood was lonely because he dreamed of becoming a painter but he failed because the adults saw his drawings in a different perspective. He was disheartened by this and so he gave up his dream. The little prince had a different problem. He left the love of his life, the rose, in his planet. When he went to Earth, he saw a garden full of roses. He felt betrayed by his love because the rose told him that it was unique and one and only rose. He was disheartened but when he met a fox who made him realize that the rose was his rose because he loves it and so it was unique from all the rest.Another important lesson that I learned from the book is that people should learn how to value friendship. Some people tend to forget some people who made an important mark in their lives. Having a true friend is rare and we all know that money cannot buy love and friendship. One of the lines in the book supports this idea. Men have no more time to understand anything. They buy things all ready made at the shops. But there is no shop anywhere where one can buy friendship, and so men have no friends anymore These close ties would make us happy because we need people who care for us and also we need to care for someone. Just like the little prince and the fox he met on earth, when the former tamed the latter, he felt responsible for the fox. When the little prince had to go already, the fox was sad because he was already attached to the prince. The little prince was unhappy because the fox wanted to cry and that he thought he only wasted the fox’s time. The fox told him that because the little prince wasted his time on him, he made the fox feel important.I learned that caring for people is essential because it would make both parties happy. The feeling that someone loves you and someone wants to be with you is a very happy feeling that no one wants to live without. As a whole, this book gave me many ideas and realizations that I would treasure in my whole life. I realized that is should not take for granted the people who loves me dearly. I learned the value of friendship and preserving them. And lastly, I learned to see things in a different perspective by looking things from my heart.

Side jobs. Bringing in cash

Side jobs. Bringing in cash High-paying side jobs You may witness people who have an insatiable desire to accomplish more every day. They are goal-oriented and ambitious, and they need an additional occupation to develop professionally and become a person they always wanted to be. Perhaps, what we said above may be attributed to you as well. In this case, this is what we have to offer in terms of job proposals. One day, you may find yourself in a tight position of a student or an office employee, who is on a limited budget or an adult, trying to pay the bills. Whenever you feel extra money will make you happier, we suggest applying for a side-job that is going to bring extra cash and professional satisfaction. Some of the most attractive options are listed below: Fitness instructor. In case you want to obtain a side hobby and earn money at the same time, becoming a fitness instructor will certainly blend in your daily routine. There are individual instructors, who are paid to train with a particular person, and there are those who are doing group classes. If you prefer to be surrounded by people, the last option will suit you well. Normally, you will be required to get a certificate for this type of job, and six months of prior trainings are compulsory to see if you fit in a specific company of instructors, depending on your ability to communicate and establish connections with people. Commonly, group classes are fun and need you to stay determined and organized, as you may have people of different ages coming to get their fair share of personal training. Your main task would be to monitor the work out process and check on the client’s progress the whole time. Archivist. Unlike the first job, this one is for people, who can spend hours, gathering information. For those, who love catalogues and works of art, being employed as an archivist is the best chance to show themselves in the professional sphere. Sometimes, your local museum would require specialists to check on the backlogs and organize papers in alphabetical order. Apparently, you will need a degree in archive management or any other discipline, closely related to the said sphere. Marketing manager. Marketing managers create plans for companies and reach out to inform clients of these plans. They can also attend the sponsorship events to see if the product has established itself among the audience, and have the responsibility of analyzing current situations in business. You will need a certain amount of qualification for this job, and you should know it will be a temporary occupation in case you don’t have experience or happen to be currently enrolled in the university. Writer. You can work as a writer with a part-time schedule, if you feel creativity is something you can’t live without. This type of profession is perfect for those, who want to study and gain experience at the same time. Besides, they pay you well if you manage to meet the deadline and provide clients with authentic, 100% original content. Working as a writer pays off in case you want to devote your life to that sphere, too, as it develops your verbal talents as well as written communication practice, which is important if you are employed by an educational institution or an essay service company. City-guide blogger. Companies and corporations love bloggers, who write articles on their local city attractions. You can be focused on anything that draws your attention and sounds entertaining, like recreation, cultural events and architecture. The key to this side-job is being able to provide sufficient information on the sightseeing tours in your area and making sure your readers enjoy the views as they collect ideas from your blog. Some real estate companies are also interested in this kind of advertising, as they work with sales and management department and need promotion for business. The vacancy is ideal for the lovers of travel and tourism – you just have to be unique and find a personal shtick that will later become your trademark. Business consultant. Business consulting represents a broad sphere of professional knowledge, but generally, you will have to talk to the business owners and provide them with valuable advice on how to improve their performance. Business consultants can make a lot of money by facilitating several companies in finding their perfect strategy and are often present at meetings, whenever administrative help is required. You have to be a good organizer with a rhetorical talent and an excellent conversationalist to maintain a level that is required for a specific business field. You also need to possess a degree in business administration and be able to handle conflict situations at work. Stress management is important for the side jobs such as this, as you never know who you are going to cooperate with.H

American History - 11 Essay Example | Topics and Well Written Essays - 1750 words - 2

American History - 11 - Essay Example In December 1965 King was chosen by NAACP to spearhead the Montgomery Bus Boycott. His inspirational oratory energized the Civil Rights Movement in Alabama. The campaign succeeded after 382 days: Montgomery repealed the law mandating segregated public transportation. In January 1957, in the aftermath of this victory, Martin Luther King Jr., Ralph Abernathy, and 60 ministers and civil rights activists founded the Southern Christian Leadership Conference, which adopted a campaign of non-violent protests to promote civil rights reform. The SCLC conducted meetings on enfranchisement and King gave nation-wide talks on racism. Inspired by Mahatma Gandhi, King made non-violent activism the keystone of his policy. In 1960, King encouraged the student â€Å"sit-in† movement in several Southern cities, and coordinated the formation of the Student Nonviolent Coordinating Committee. King was now a national celebrity and the symbol of the Civil Rights Movement.   On August 28, 1963, King led the mammoth ‘March on Washington’ which drew more than 200,000 people to the Lincoln Memorial. It was here that King made his famous â€Å"I Have a Dream† speech, asserting his belief in the brotherhood of all men. He played a pivotal role in the passage of the Civil Rights Act of 1964 mandating desegregation of public accommodations and in publicly owned facilities and the Voting Rights Act of 1965.He received the Nobel Peace Prize for 1964. He was assassinated by  James Earl Ray, a former convict, in Memphis on April 4, 1968. Martin Luther King Jr. may be considered one of the greatest personalities who shaped the American Civil Rights Movement. 1. 27-3 Brown vs Board of Education of Topeka served as the turning point in the constitutional battle against segregation. Plessy v. Ferguson  (1896) legalized separate but equal school systems for blacks and whites. By rejecting the contention of the School Board that the