2. Cell fusion
In this step HGPRT defective myeloma cells are mixed with thoroughly clean lymphocytes. The mixture of the cells are then exposed to a strong concentration of PEG and fusion is allowed to occur. The PGE ejected by washing and the cells are maintained In a well nurse medium. Three types of cells are found in the medium. They are –

? mixture of Hybridomas
?free myeloma cells
?free lymphocytes

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3. Selection
For selection of hybrid cells hypoxanthine aminopterin thymidine medium (HAT) is used. In HAT medium the cellular synthesis of purines and pyrimidines from simple sugar is blocked by aminopterin. But some cells can flourish by taking advantage of hypoxanthine and thymidine existent in the medium by salvage pathway utilizing hypoxanthine guanine phosphoribosyl transferase (HGPRT). But HGPRT is deficient in myeloma cells. So when aminopterin block denovo pathway myeloma cells can not survive in HAY medium. Beta cell can survive in the HAT medium as they are HGPRT+ . Beta cell go through natural cell death following some division.

4. Screening
For the secretion of the antibody of aimed specificity, the Hybridomas must be screened. For the aimed antibody specificity the culture from individual Hybridoma culture is being tested periodically. ELIAS and RIA are mostly used technique for this reason. In these experiments, the antibody is attached to the specific antigen and unwind antibody and remaining components of the medium can be sweeped away. By this way, using screening we can identify the Hybridoma cells which can produces desired antibody. The antibody produce by the hybrid cells are monoclonal antibody.
5. Cloning and propagation
The individual Hybrid cells which produce the intended antibody are isolated and cloned. For cloning hybrid cells two methods are usually used.
Limiting dilution method: In this method, gradual dilution of the suspension of Hybridoma cells is made and aliquots of individual dilution are place down into micro culture wall. The dilution are made to point that every aliquot in a wall contains just one individual hybrid cell. This guarantees the immunglobin which has delivered is monoclonal.
Soft agar method: In this procedure the Hybridoma cells are refined in soft agar. It is conceivable to develop numerous cells at the same time in semisolid medium to form colonies. These colonies will be monoclonal in nature.

In genuine practice both above system are consolidated and utilized for the maximum production of MAbs.
6. Characterization and storage :
The monoclonal antibody must be exposed to biochemical and biophysical portrayal for the intended specificity. It is likewise vital to clarify the MAbs for the immunoglobulin class or sub-class, the epitope for which it is particular and quantity of binding site it has . The strength of the cell lines and the MAbs are essential. The cells must be described for their capacity to withstand solidifying and defrosting.

2.6 GENOME PLASTICITY AND EVOLUTION OF ESCHERICHIA COLI
Like all life forms, new strains of E. coli evolve through the natural biological processes of mutation, gene duplication, and horizontal gene transfer; in particular, 18% of the genome of the laboratory strain MG1655 was horizontally acquired since the divergence from Salmonella. E. coli K-12 and E. coli B strains are the most frequently used varieties for laboratory purposes. Some strains develop traits that can be harmful to a host animal. These virulent strains typically cause a bout of diarrhea that is often self-limiting in healthy adults but is frequently lethal to children in the developing world. (Futadar et al., 2005). More virulent strains, such as O157:H7, cause serious illness or death in the elderly, the very young, or the immunocompromised.
The genera Escherichia and Salmonella diverged around 102 million years ago (credibility interval: 57–176 mya), which coincides with the divergence of their hosts: the former being found in mammals and the latter in birds and reptiles. (Wang et al., 2009). This was followed by a split of an Escherichia ancestor into five species (E. albertii, E. coli, E. fergusonii, E. hermannii, and E. vulneris). The last E. coli ancestor split between 20 and 30 million years ago.
The long-term evolution experiments using E. coli, begun by Richard Lenski in 1988, have allowed direct observation of genome evolution over more than 65,000 generations in the laboratory. For instance, E. coli typically do not have the ability to grow aerobically with citrate as a carbon source, which is used as a diagnostic criterion with which to differentiate E. coli from other, closely, related bacteria such as Salmonella. In this experiment, one population of E. coli unexpectedly evolved the ability to aerobically metabolize citrate, a major evolutionary shift with some hallmarks of microbial speciation.
2.7 INCUBATION PERIOD
The time between ingesting the STEC bacteria and feeling sick is called the “incubation period”. The incubation period is usually 3–4 days after the exposure, but may be as short as 1 day or as long as 10 days. The symptoms often begin slowly with mild belly pain or non-bloody diarrhea that worsens over several days. HUS, if it occurs, develops an average of 7 days after the first symptoms, when the diarrhea is improving.

2.7.1 DISCOVERY OF ANTIBIOTICS
• History of antibiotics – 1
19th century:Louis Pasteur & Robert Koch
• History of antibiotics – 2
Plant extracts
– Quinine (against malaria)
– Ipecacuanha root (emetic, e.g. in dysentery)
Toxic metals
– Mercury (against syphilis)
– Arsenic (Atoxyl, against Trypanosoma)
• Dyes
– Trypan Blue (Ehrlich)
– Prontosil (azo-dye, Domagk, 1936)
• History of antibiotics – 3
Paul Ehrlich
• started science of chemotherapy
• Systematic chemical modifications
(“Magic Bullet”) no. 606 compound = Salvarsan (1910)
• Selective toxicity.
• Developed the Chemotherapeutic Index
• History of antibiotics – 4
Penicillin- the first antibiotic – 1928• Alexander Fleming observed the
killing of staphylococci by a fungus (Penicillium notatum)
• observed by others – never exploited
• Florey & Chain purified it by freeze-drying (1940) – Nobel prize 1945
• First used in a patient: 1942
• World War II: penicillin saved 12-15% of lives
• History of antibiotics – 5
Selman Waksman – Streptomycin (1943), was the first scientist who discovered antibiotic active against all Gram-negatives for examples; Mycobacterium tuberculosis
– Most severe infections were caused by Gram-negatives and Mycobacterium
tuberculosis, extracted from Streptomyces – extracted from Streptomyces
– 20 other antibiotics include. neomycin, actinomycin
2.8 CHARACTERISTICS OF ANTIBIOTICS
According to the Oxford Dictionary, the term Antibiotics encompasses medicines (such as penicillin or its derivatives) that inhibit the growth of or destroys microorganisms. Antibiotics are naturally occurring substances that exhibit inhibitory properties towards microbial growth at high concentrations. (Zaffiri, et al., 2012).
-Antibiotics are selective in their effect on different microorganisms, being specific in their action not only against genera and species but even against strains and individual cells. Some of these agents act mainly on gram-positive bacteria, while others inhibit only gram-negative ones.
-Some antibiotics are produced by some organism, from different strains of penicillin.
-Bacteria are sensitive to the antibiotic which enable them to developed resistance after contact, for several periods.

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2.9 ROLE OF ANTIBIOTICS
Based on the clinical use of antibiotics, it may appear that these compounds play a similar role as microbial weapons in nature, yet this seems unlikely due to the fact that the concentrations used in the clinical setting are significantly higher than that produced in nature (Fajardo et al., 2008). Due to experimental evidence, it makes more sense to see antibiotics as small, secreted molecules involved in cell-to-cell communication within microbial communities.
(Martinez, 2008). Diverse Studies have been conducted in which different antibiotics and antibiotic-like structures were administered to different bacterial species at levels below the compounds minimum inhibitory concentrations (MIC). (Fajardo et al., 2008). that was

2.1.1 HISTORY OF PSEUDOMONAS AERUGENOSA
Pseudomonas aerogenosa was first recognized in the study “On the blue and green coloration of bandages” in 1882, conducted by Carle Gessard a French pharmacist. In his study he discovered that P.aerugenosa was a water-soluble pigment, which under exposure to ultraviolet light, illuminated green-blue. Carle Gessard, back in 1882, concluded that P.aerugenosa was of a pathogenic, infectious nature, after classifying the strand; due to the similarity between the strand and other similar microbes.
Since the discovery of this opportunistic pathogen, breakthroughs have been made, sighting the severity of its power to fester rapidly and oppose treatment. This pathogen is constantly monitored, and its genome is continually updated into data bases, due to the potential for it to be used as a biological weapon
2.1.1.1 HISTORY OF ESCHERICHIA COLI
In 1885, the German-Austrian pediatrician Escherichia disc overfed this organism in the feces of healthy individuals. He called it Bacterium coli commune because it is found in the colon. Early classifications of prokaryotes placed these in a handful of genera based on their shape and motility (at that time Ernst Haeckel’s classification of bacteria in the kingdom Monera was in place).Bacterium coli was the type species of the now invalid genus Bacterium when it was revealed that the former type species (“Bacterium triloculare”) was missing. Following a revision of Bacterium, it was reclassified as Bacillus coli by Migula in 1895 and later reclassified in the newly created genus Escherichia, named after its original discoverer. Bacterium coli has since been used for biological lab experiment research, infection can lead to hemolytic uremic syndrome (HUS), characterized by hemolytic anemia, thrombocytopenia, and renal injury.1996 the world’s worst outbreak of E. coli food poisoning occurred in Wishaw, Scotland, killing 20 people.

2.1.1.2 EPIDEMIOLOGY AND CONTROL OF PSEUDOMONAS AERUGENOSA
P. aeruginosa is primarily a nosocomial pathogen, and the methods for control of infection are similar to those for other nosocomial pathogens. Because pseudomonas thrives in moist environments, special attention should be paid to sinks, water baths, showers, hot tubs, and other wet areas. For epidemiologic purposes, strains can be typed using molecular typing techniques.
2.1.1.3 EPIDEMIOLOGY PREVENTION AND CONTROL OF ESCHERICHIA COLI
The enteric bacteria establish themselves in the normal intestinal tract within a few days after birth and from then on constitute a main portion of the normal aerobic (facultative anaerobic) ssmicrobial flora. E. coli is the prototype. Enteric that are mostly found in water found in water or milk are accepted as proof of fecal contamination from sewage or other sources.
Control measures are not feasible as far as the normal endogenous flora is concerned. Enteropathogenic E .coli serotypes should be controlled like salmonellae. Some of the enteric constitute a major problem in hospital infection. It is particularly important to recognize that many enteric bacteria are “opportunists” that cause illness when they are introduced into debilitated patients. Within hospitals or other institutions, these bacteria commonly are trans-mitted by personnel, instruments, or parenteral medications. Their control depends on hand washing, rigorous asepsis, sterilization of equipment, disinfection, restraint in intravenous therapy, and strict precautions in keeping the urinary tract sterile (i.e. closed drainage).

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2.1.1.4. MICROBIOLOGY AND DIAGNOSIS OF PSEUDOMONAS AERUGENOSA
A. Specimens
Specimens from skin lesions, pus, urine, blood, spinal fluid, sputum, and other material should be obtained as indicated by the type of infection.
B. Smears
Gram-negative rods are often seen in smears. No specific morphologic characteristics differentiate pseudomonads in specimens from enteric or other gram-negative rods.
C. Culture
Specimens are plated on blood agar and the differential media commonly used to grow the enteric gram-negative rods. Pseudomonads grow readily on most of these media, but they may grow more slowly than the enteric. P. aeruginosa does not ferment lactose and is easily differentiated from the lactose-fermenting bacteria. Culture is the specific test for diagnosis of P aeruginosa infection. (Henry et al,. 2011).

2.2. PATHOGENESIS OF PSEUDOMONAS
P. aeruginosa is pathogenic only when introduced into areas devoid of normal defenses, such as when mucous membranes and skin are disrupted by direct tissue damage as in the case of burn wounds; when intravenous or urinary catheters are used; or when neutropenia is present, as in cancer chemo-therapy. The bacterium attaches to and colonizes the mucous membranes or skin, invades locally, and produces systemic disease. These processes are promoted by the pili, enzymes, and toxins described earlier. Lipopolysaccharide plays a direct role in causing fever, shock, oliguria, leukocytosis and leukopenia, disseminated intravascular coagulation, and adult respiratory distress syndrome.
P. aeruginosa and other pseudomonads are resistant to many antimicrobial agents and therefore become dominant and important when more susceptible bacteria of the normal microbiota are suppressed. May lead to rapid destruction of the eye, occurs most commonly after injury or surgical procedures. In infants or debilitated persons, P .aeruginosa may invade the bloodstream and result in fatal sepsis; this occurs commonly in patients with leukemia or lymphoma who have received antineoplastic drugs or radiation therapy and in patients with severe burns.
2.2.1 PATHOGENESIS AND CLINICAL MANIFESTATION OF ESCHERICHIA COLI
The clinical manifestations of infections with E coli and the other enteric bacteria depend on the site of the infection and cannot be differentiated by symptoms or signs from processes caused by other bacteria.
A. E coli
1. Urinary tract infection—E coli is the most common cause of urinary tract infection and accounts for approximately 90% of first urinary tract infections in young women. The symptoms and signs include urinary frequency, dysuria, hematuria, and pyuria. Flank pain is associated with upper tract infection. None of these symptoms or signs is specific for E. coli infection. Urinary tract infection can result in bacteremia with clinical signs of sepsis. Most of the urinary tract infections that involve the bladder or kidney in an otherwise healthy host are caused by a small number of O antigen types that have specifically elaborated virulence factors that facilitate colonization and sub-sequent clinical infections. These organisms are designated as uropathogenic E coli. Typically, these organisms produce hemolysin, which is

2. Christmas is celebrated as the birthday of Jesus Christ but there are no records as 25th December is the birthday of Jesus.
3. 25th December was chosen because it coincides with pagan festival Saturnalia.
4. Christmas was first celebrated in 336.
5. The first artificial Christmas tree was made in the 19th century in Germany.
6. In Boston, from 1659 to 1681 it was prohibited to celebrate Christmas.
7. Firstly, Santa Claus was the short name given to the Christian Bishop of Turkey, St. Nicholas. He was wealthy and used to help the needy people and also became the protector of children.
8. Christmas is sometimes written as Xmas; X in Greek abbreviates for Christ.
9. In mid-17th century Christmas trees were decorated with candles.
10. Christmas trees were also decorated with candies, small gifts, cake, nuts, and fruits.
11. The tallest artificial Christmas tree is in Sri Lanka, it is 72.1 m.
12. The tallest real Christmas tree was 67.36 meters and was in Seattle, Washington.
13. The first artificial Christmas trees were made by Germans.
14. Christmas pudding is the main dish of Christmas.
15. The highest-grossing Christmas movie is Jim Carrey ‘How The Grinch Stole Christmas’.
16. in Canada, there is official address of Santa Claus- North Pole, H0H 0H0, Canada.
17. Approximately 350 million Christmas trees growing in the U.S. farms.
18. Before selling Christmas tree it is grown for 7-10 years.
19. Since 1850 Christmas trees were sold in the US.
20. Until June 26, 1870, Christmas was not the official holiday in the US.
21. Franklin Pierce was the first US president to put Christmas tree in White House.
22. The tradition of Christmas cards is originated in England.
23. The most expensive Christmas card was sold in the auction for £20,000 in the UK.
24. Santa Claus is known by different names around the world- in Russia Deushka Moroz, in Germany Kriss Kringle, etc.
25. Since 1960, half of the population Sweden still watch Donald Duck cartoon.
26. Jingles Bells was originally written for Thanksgiving, not for Christmas.
27. Red and white colored dress Santa Claus was first used by Coca-Cola for promotions.
28. People of Iceland celebrate Christmas Eve by exchanging books and spending rest of the night reading it and eating chocolates.
29. Japanese go to KFC for dinner on Christmas.
30. 230,000 tons of Christmas food is wasted every year.
31. 10 million turkeys are cooked on Christmas in the UK.
32. Burning Yule logs on Christmas is a symbol of luck, health and fertility, and also a guard against evil spirits.
33. Evergreen trees are a symbol of rebirth from the time of paganism.
34. In 2012, 30,000 people participated in Secret Santa organized by Reddit.
35. At the Christmas in Finland, it is customary to go to the sauna.
36. 6.8 million Apple and Android device are bought on the Christmas.
37. French gifted Statue of Liberty to the US on Christmas.
38. In honor of Christmas in Spain, the largest lottery is held annually.
39. In Germany and England, the main dish of the Christmas table is a roast goose or duck.
40. On the eve of the Christmas, Lithuanian Catholics eat only lean food
41. In Russia, Christmas celebration started in the 20th century.
42. The first song for Christmas was written in the 4th century AD.
43. In 2010, a Facebook study showed that 2 weeks before the Christmas is the time when most of the breakups happen.
44. The European live Christmas tree demand reaches about 50 million every year.
45. Oregon, North Carolina, Michigan, Pennsylvania, Wisconsin, and Washington are the most Christmas tree producing states.
46. The tradition of filling stockings with gifts on a holiday came from the story of three poor sisters. The legend says that one day St Nicholas made his way to them through the chimney and left gold coins in stockings.
47. The first Christmas cracker was invented in 1847 by sweets vendor Tom Smith
48. Charles Dicken wrote Christmas Carols in just six weeks.
49. Every year 1.6 billion Christmas Cards are purchased by Americans.
50. The first Christmas card was created by the Englishman Henry Cole in 1843.
51. In 1810, in the US, the public first saw Santa Claus.
52. The first case of the gift was noted in ancient Rome, where on the celebration of Saturnalia, children were given gifts.
53. orthodox Christian celebrate Christmas on January 7.
54. Traditional colors for decorating a Christmas tree are green, red and gold.
55. the first street Christmas trees with electric garlands appeared in Finland in 1906.
56. In Poland, spiders or spider webs are the usual decoration of the Christmas tree, because according to legend, the spider wove a blanket for Jesus when he was an infant.
57. The British wear paper crowns for the Christmas dinner. Crowns are stored in a special tube, which is called “Christmas cracker”.
58. On Christmas Eve in 1914, warring parties on the Western Front of the First World War stopped shooting and sang Christmas carols, and the next morning German and British soldiers even exchanged handshakes and gifts.
59. In the Netherlands, on Christmas night, children put a shoe to the fireplace for gifts and put a carrot for a magic horse.

The Alexandrian theologians proposed to celebrate Christmas on May 26 in 200 BC.

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2) To what extent, if at all, was it possible to roll back the French Revolution with the Congress of Vienna? Was a complete rollback even desirable? Why or why not?
1. Introduction
Whether a complete rollback of the French Revolution (Revolution) was desirable yields no simple answer, but it is apparent that the Congress of Vienna had rolled back the doings of the French Revolution in form but not in substance. To that end, this paper will first discuss what the congressmen stood for mainly between 1815 and 1848 in response to the legacies left behind after the era of the Revolution; before scrutinising the success and appeal, or lack thereof, for a complete rollback.
2. Conservative Backlash
(Superficial) Restoration of the Political Regime
Following the unrest of the French Revolution was the period of 1815-1848, or the “Era of Metternich”, seeking to restore the balance of power in Europe that was tipped over during the French Revolution. One of reaction, the alliance amongst Austria, Britain, Prussia, Russia, led by Austrian Chancellor Metternich, saw the need for a general reconciliation of European affairs and Napoleon’s legacy. With that came along a redrawing of the map of Europe, the restoration of old rulers and the reassignment of territories as established by the Congress of Vienna in 1815. Prussia and Austria, for example, expanded their existing territories with the reassignment, even Poland became part of Russia; and in France, the Bourbon monarch was restored as “legitimate” rulers of the French since 1792 to undo the dynastic changes introduced by Napoleon and suppress the rising liberal, nationalist rebels that favoured greater political, intellectual and economic freedom for the individual. To preserve these resettlements, the Concert of Europe, a series of conferences attended by the Crown heads of Europe, served as a platform for the rulers to solve political problems diplomatically before they grew out of proportion. There is no doubt that the age of Metternich, characterised by support for political absolutism, suppression of nationalistic ambitions and forcible preservation of the status quo, kept Europe in check through collective effort and systematic changes, albeit forceful.
No Restoration of Ideals
However, the efforts of the Congress of Vienna did not roll back the Revolution insofar as it was apparent that the revolutionary nationalist sentiments had already been sown throughout Europe. While the period of 1815-1848 saw Europe in a period of temporary peace, it is not to say that it was a time free of revolutionary stirrings and sentiments in support of nationalistic ideals. This is especially evident by the stirrings of Revolution in 1830 and the full scale revolutions of 1848. Despite successful revolutions that led to the independence of Greece and Belgium in 1830, similar unrest persisted in the same year with the 1830 French Revolution after Louis XVIII promulgated a Charter that made France a constitutional monarchy and accepted equality before the law and the Napoleonic Code. His successor, Charles X, on the other hand was a staunch reactionary who sought to roll back the gains of the Revolution and moved towards a more absolutist regime once again. By 1830, discontentment with reaction and suppression of nationalistic ambitions had reached explosive proportions: Charles X published his July Ordinance restricting further the freedom of the press; dissolving the new Chamber to which a liberal majority had been elected; promulgating a new electoral law narrowing the suffrage, and calling for new elections which would presumably result in the return of a reactionary majority. This escalated to a vote of no confidence and his abdication, bypassing the “legitimate” Bourbon line with the Duke of Orleans and laid the grounds for the 1848 Revolution, a continent-wide revolution most notably arising in France, the Italian and German states, Hungary. Indeed, stirrings and unrest following 1830 saw the propagation of the seeds of nationalism amongst people, especially the middle class, who were evidently pushing back against a more absolutist regime envisioned by the congress. They were a force to be reckoned with but obviously overlooked by the Congress of Vienna.
3. Appeal of the Congress of Vienna
While Metternich and other rulers believed in the power of the congress to establish peace and order in Europe, particularly where its legislative sovereigns and an international balance of power could not be challenged by the forces of liberalism and nationalism or a single state, the same vision was not shared by the emerging bourgeoisie who favoured exactly the opposite. The French Revolution and the Napoleonic Wars unleashed forces that shook the foundations of European society. Napoleon had spread ideas of democracy, liberty and equality, and planted the seeds of representative government across Europe. Metternich had retarded liberalism but had failed to eradicate the influence of the French Revolution, especially in the midst of unrest and conflict that created further discontentment at the bottom of the regime.
5. Conclusion
The Congress of Vienna was organised with good intentions of preserving peace in Europe, albeit a reactionary one. While no major conflict was realised, there was clearly a great deal of unrest, especially amongst the emerging middle class that was disenfranchised and thus repulsed the Congress and the old order. Given that a backward change in political form could not contain, let alone undo, people’s shifting attitudes towards political liberation, rolling back the gains of the Revolution with the Congress of Vienna was possible at a superficial level and to a small extent, and only desirable in the eyes of the conservatives and not that of the people.
(917 words)

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