Effect of antibiotics on growth curve by using micro organisams

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this anil iam b tech final year biotechnology from sri indu college of engg and technology effect of antibiotics on growth curve o

Effect of antibiotics on growth curve by using micro organisms

PROJECT REPORT ON “EFFECT OF ANTIBIOTICS ON GROWTH CURVE BY USING MICRO ORGANISAMS” Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of BACHELOR OF TECHNOLOGY
In BIOTECHNOLOGY By

CHALLAGUNDLA ANIL (08D41A2351)

THEERTHALA VEN (08D41A2359)

Under the Guidance of

MS. Dr. B. SUNITHA Head of Department BIOTECHNOLOGY

DEPARTMENT OF BIOTECHNOLOGY SRI INDU COLLEGEOF ENGINEERING AND TECHNOLOGY Sheriguda vil, Ibrahimpatnam, RR Dist – 501510 ( Affiliated to J.N.T.University, Kukatpally, A.P, INDIA ) 2012

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Effect of antibiotics on growth curve by using micro organisms

CONTENTS 1. Abstract

PAGE No.

2. Introduction………………………………………………………..

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3. Bacteria of E. coli…………………………………………………. 3 4. Role of Antibiotics…………………………………………………. 6 5. Bacterial mechanisms of antibiotic resistance ……………………….7 6. Antibiotics List & Classification…………………………………….. 12 1. 2. 3. 4. Ampicillin Penicillin Tetracycline Streptomycin

7. Materials of using for effect of antibiotics……………………………. 29 8.1 Autoclave 8.2 Laminar Air Flow 8.3 Incubator 8.4 Nephelometer 8.5 UV Spectrophotometer 9. Methodology…………………………………………………….. 9.1 Preparation Of Nutrient Agar……………………………………….. 44 9.2 Preparation of antibiotic stock solutions………………………………. 46 Sri indu college of engineering and technology | page no 2

Effect of antibiotics on growth curve by using micro organisms

9.3 Preparation of dried filter paper Antibiotics discs………………………… 48

9.4 Storage of commercial antimicrobial discs………………………….. 49 9.5 Preparation of E. coli Culture…………………………………….. 50 9.6 Preparation of inoculums………………………………………….. 51 9.7 Inoculation of the nutrient Agar plates……………………………….52 10. Antibiotic Sensitivity Tests…………………………………………… 54 11. Incubation of the plates………………………………………………. 56 12. Measuring zone sizes……………………………………………………56 13. Result &Interpretation………………………………………………… 57 13.1 Antibiotic discs plates………………………………………………… 58-61 13. 2 Tables& graphs………………………………………………………62-101 13.3 Conclusion……………………………………………………………. 102 14. Selected Bibliography……………………………………………………… 103

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Effect of antibiotics on growth curve by using micro organisms

Abstract:
The effect of antibiotics on growth curve by using Microorganisms characteristics, the antibiotic sensitivity and the ultra structure of E. coli bacteria cells have been studied. Equal volumes of E. coli cells were exposed to the magnetic field for different periods, the two most effective periods, namely, 6 h and 16 h were chosen for all our experimental studies. The results indicated that exposure of the microorganisms to the demonstrated magnetic field caused pronounced changes in the growth characteristic curves, where a suppressive effect was observed on the cell growth and the number of cells at stationary phase markedly decreased after exposure period of 6 h but there was a slight increase in the growth rate after exposure period of 16 h with increase in the number of cells. Further, changes in the antibiotic sensitivity was observed after exposure period of 6 h since E. coli cells became more sensitive to certain antibiotics such as ampcilin, penicillin, and tetracycline, streptomycin as revealed in the increase in their zone diameters while, after a 16 h exposure period, it became more resistant to the same antibiotics. Furthermore, the results of the ultra structure showed that while exposure period 6 h decreased the cell length, the exposure period 16 h elongated the cell length with decreasing the thickness of the cell wall beside the disappearance of the majority of cytoplasm components. The effect of antibiotic resistance and antibiotic sensitivity and zone of inhibition studied for Nefhelometer and UV Spectrophotometer

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Effect of antibiotics on growth curve by using micro organisms

2.Introduction:
Health is very important to human survival and happiness. The world’s smallest living organisms, bacteria, sometimes can threaten human health. Bacteria are everywhere, inside and outside the human body. Some bacteria make people sick, and in some cases can be fatal.

3.Bacteria Of E. coli:
Bacteria are simple organisms that consist of one cell lacking a nucleus. They are among the smallest living things on earth. Most bacteria measure from 0.2 to 0.3 microns. The diameter can only be seen through a microscope. Scientists classify bacteria as prokaryotes. Bacteria are widespread, present in soil, air, and water, and as parasites on and in other living things. Bacteria were likely the only form of life on earth for over two billion years. Antony van Leeuwenhoek first observed them in the 17th century. Bacteriology as an applied science began to develop in the 19th century. Bacteria are amazingly adaptable to varied environmental conditions. They are found in the bodies of all living organisms and on all parts of the earth and ocean depths, artic ice and glaciers, in hot springs, and even in the stratosphere. The understanding of bacteria and their metabolic processes have been expanded by the discovery of species that can live only deep below the earth’s surface and by species that thrive without sunlight in the high temperature and pressure near hydrothermal vents on the ocean floor. There are more bacteria, as separate individuals, than any other type of organism. There can be as many as 2.5 billion bacteria in one gram of fertile soil. Characteristics Bacteria are grouped in a number of different ways. Most bacteria are of one of three typical shapes: rod- shaped (bacillus), round (cocas, e.g., streptococcus), and spiral (spirillum). Another group, vibrios, appears as incomplete spirals. The cytoplasm and plasma membrane of most bacterial cells are surrounded by a cell wall. The further classification of bacteria is based on cell wall characteristics. They can also be characterized by their patterns of growth, such as the chains formed by streptococci. Many bacteria, primarily the bacillus and spiral forms, are motile,

swimming about by whip like movements of flagella; other bacteria have rigid rod like protuberances called "pile" that is serve as tethers. Some bacteria can function only in the presence of oxygen. Others cannot grow in the presence of free oxygen but obtain oxygen from compounds. Facultative anaerobes can grow with or without free oxygen; obligate anaerobes are poisoned by oxygen

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Pathogenic Bacteria Bacteria that cause disease are called pathogens. Among bacterial plant diseases are leaf spot, fire blight and wilts. Animal diseases caused by bacteria include tuberculosis, cholera, syphilis, typhoid fever, and tetanus. Some bacteria attack the tissues directly; others produce poisonous substances called toxins. Antibodies provide natural defense against harmful bacteria. Certain bacterial diseases, e.g., tetanus, can be prevented by injection of antitoxin or of serum containing antibodies against specific bacterial antigens; immunity to some can be induced by vaccination; and certain specific bacterial parasites are killed by antibiotics. New strains of more virulent bacterial pathogens, which many resistant to antibiotics, have appeared in recent years. Many believe this is due to the overuse antibiotics, which are both prescriptions of minor ailments. Helpful Bacteria Certain types of bacteria live in the intestines of humans and animals. These bacteria help in digestion and in destroying harmful organisms. Intestinal bacteria also produce some vitamins needed to help the human body. Bacteria in soil and water play a vital role in recycling carbon, nitrogen, sulfur, and other chemical elements used by living things. Many bacteria help decompose dead organisms and animal wastes into chemical elements. Certain kinds of bacteria invert.

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Organism:

(E.coli structure)

Bacteria Cell Structure They are as unrelated to human beings as living things can be, but bacteria are essential to human life and life on planet Earth. Although they are notorious for their role in causing human diseases, from tooth decay to the Black Plague, there are beneficial species that are essential to good health.

For example, one species that lives symbiotically in the large intestine manufactures vitamin K, an essential blood clotting factor. Other species are beneficial indirectly. Bacteria give yogurt its tangy flavor and sourdough bread its sour taste. They make it possible for ruminant animals (cows, sheep, goats) to digest plant cellulose and for some plants, (soybean, peas, alfalfa) to convert nitrogen to a more usable form.

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Bacteria are prokaryotes, lacking well-defined nuclei and membrane-bound organelles, and with chromosomes composed of a single closed DNA circle. They come in many shapes and sizes, from minute spheres, cylinders and spiral threads, to flagellated rods, and filamentous chains. They are found practically everywhere on Earth and live in some of the most unusual and seemingly inhospitable places. Evidence shows that bacteria were in existence as long as 3.5 billion years ago, making them one of the oldest living organisms on the Earth. Even older than the bacteria are the archeans (also called archaebacteria) tiny prokaryotic organisms that live only in extreme environments: boiling water, supersalty pools, sulfur-spewing volcanic vents, acidic water, and deep in the Antarctic ice. Many scientists now believe that the archaea and bacteria developed separately from a common ancestor nearly four billion years ago. Millions of years later, the ancestors of today's eukaryotes split off from the archaea. Despite the superficial resemblance to bacteria, biochemically and genetically, the archea are as different from bacteria as bacteria are from humans. In the late 1600s, Antoni van Leeuwenhoek became the first to study bacteria under the microscope. During the nineteenth century, the French scientist Louis Pasteur and the German physician Robert Koch demonstrated the role of bacteria as pathogens (causing disease). The twentieth century saw numerous advances in bacteriology, indicating their diversity, ancient lineage, and general importance. Most notably, a number of scientists around the world made contributions to the field of microbial ecology, showing that bacteria were essential to food webs and for the overall health of the Earth's ecosystems. The discovery that some bacteria produced compounds lethal to other bacteria led to the development of antibiotics, which revolutionized the field of medicine. There are two different ways of grouping bacteria. They can be divided into three types based on their response to gaseous oxygen. Aerobic bacteria require oxygen for their health and existence and will die without it. Anerobic bacteria can't tolerate gaseous oxygen at all and die when exposed to it. Facultative aneraobes prefer oxygen, but can live without it. The second way of grouping them is by how they obtain their energy. Bacteria that have to consume and break down complex organic compounds are heterotrophs. This includes species that are found in decaying material as well as those that utilize fermentation or respiration. Bacteria that create their own energy, fueled by light or through chemical reactions, are autotrophs.
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Capsule - Some species of bacteria have a third protective covering, a capsule made up of polysaccharides (complex carbohydrates). Capsules play a number of roles, but the most important are to keep the bacterium from drying out and to protect it from phagocytosis (engulfing) by larger microorganisms. The capsule is a major virulence factor in the major disease-causing bacteria, such as Escherichia coli and Streptococcus pneumoniae. Nonencapsulated mutants of these organisms are avirulent, i.e. they don't cause disease. Cell Envelope - The cell envelope is made up of two to three layers: the interior cytoplasm membrane, the cell wall, and -- in some species of bacteria -- an outer capsule. Cell Wall - Each bacterium is enclosed by a rigid cell wall composed of peptidoglycan, a proteinsugar (polysaccharide) molecule. The wall gives the cell its shape and surrounds the cytoplasmic membrane, protecting it from the environment. It also helps to anchor appendages like the pili and flagella, which originate in the cytoplasm membrane and protrude through the wall to the outside. The strength of the wall is responsible for keeping the cell from bursting when there are large differences in osmotic pressure between the cytoplasm and the environment.

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Cell wall composition varies widely amongst bacteria and is one of the most important factors in bacterial species analysis and differentiation. For example, a relatively thick, meshlike structure that makes it possible to distinguish two basic types of bacteria. A technique devised by Danish physician Hans Christian Gram in 1884, uses a staining and washing technique to differentiate between the two forms. When exposed to a gram stain, gram-positive bacteria retain the purple color of the stain because the structure of their cell walls traps the dye. In gram-negative bacteria, the cell wall is thin and releases the dye readily when washed with an alcohol or acetone solution.
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Cytoplasm - The cytoplasm, or protoplasm, of bacterial cells is where the functions for cell growth, metabolism, and replication are carried out. It is a gel-like matrix composed of water, enzymes, nutrients, wastes, and gases and contains cell structures such as ribosomes, a chromosome, and plasmids. The cell envelope encases the cytoplasm and all its components. Unlike the eukaryotic (true) cells, bacteria do not have a membrane enclosed nucleus. The chromosome, a single, continuous strand of DNA, is localized, but not contained, in a region of the cell called the nucleoid. All the other cellular components are scattered throughout the cytoplasm. One of those components, plasmids, are small, extrachromosomal genetic structures carried by many strains of bacteria. Like the chromosome, plasmids are made of a circular piece of DNA. Unlike the chromosome, they are not involved in reproduction. Only the chromosome has the genetic instructions for initiating and carrying out cell division, or binary fission, the primary means of reproduction in bacteria. Plasmids replicate independently of the chromosome and, while not essential for survival, appear to give bacteria a selective advantage. Plasmids are passed on to other bacteria through two means. For most plasmid types, copies in the cytoplasm are passed on to daughter cells during binary fission. Other types of plasmids, however, form a tubelike structure at the surface called a pilus that passes copies of the plasmid to other bacteria during conjugation, a process by which bacteria exchange genetic information. Plasmids have been shown to be instrumental in the transmission of special properties, such as antibiotic drug resistance, resistance to heavy metals, and virulence factors necessary for infection of animal or plant hosts. The ability to insert specific genes into plasmids have made them extremely useful tools in the fields of molecular biology and genetics, specifically in the area of genetic engineering.

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Cytoplasmic Membrane - A layer of phospholipids and proteins, called the cytoplasmic membrane, encloses the interior of the bacterium, regulating the flow of materials in and out of the cell. This is a structural trait bacteria share with all other living cells; a barrier that allows them to selectively interact with their environment. Membranes are highly organized and asymmetric having two sides, each side with a different surface and different functions. Membranes are also dynamic, constantly adapting to different conditions. Flagella - Flagella (singular, flagellum) are hairlike structures that provide a means of locomotion for those bacteria that have them. They can be found at either or both ends of a bacterium or all over its surface. The flagella beat in a propeller-like motion to help the bacterium move toward nutrients; away from toxic chemicals; or, in the case of the photosynthetic cyanobacteria; toward the light. Nucleoid - The nucleoid is a region of cytoplasm where the chromosomal DNA is located. It is not a membrane bound nucleus, but simply an area of the cytoplasm where the strands of DNA are found. Most bacteria have a single, circular chromosome that is responsible for replication,

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Effect of antibiotics on growth curve by using micro organisms

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although a few species do have two or more. Smaller circular auxiliary DNA strands, called plasmids, are also found in the cytoplasm. Pili - Many species of bacteria have pili (singular, pilus), small hairlike projections emerging from the outside cell surface. These outgrowths assist the bacteria in attaching to other cells and surfaces, such as teeth, intestines, and rocks. Without pili, many disease-causing bacteria lose their ability to infect because they're unable to attach to host tissue. Specialized pili are used for conjugation, during which two bacteria exchange fragments of plasmid DNA. Ribosomes - Ribosomes are microscopic "factories" found in all cells, including bacteria. They translate the genetic code from the molecular language of nucleic acid to that of amino acids—the building blocks of proteins. Proteins are the molecules that perform all the functions of cells and living organisms. Bacterial ribosomes are similar to those of eukaryotes, but are smaller and have a slightly different composition and molecular structure. Bacterial ribosomes are never bound to other organelles as they sometimes are (bound to the endoplasmic reticulum) in eukaryotes, but are free-standing structures distributed throughout the cytoplasm. There are sufficient differences between bacterial ribosomes and eukaryotic ribosomes that some antibiotics will inhibit the functioning of bacterial ribosomes, but not a eukaryote's, thus killing bacteria but not the eukaryotic organisms they are infecting.

4. Role of antibiotics:
Antibiotics are body compounds used to inhibit bacteria growth. In the beginning antibiotic referred only to natural compounds. They are produced by bacteria. Penicillin is the most familiar antibiotic and has been used to fight many infectious diseases including syphilis, gonorrhea, tetanus, and scarlet fever. Another antibiotic, streptomycin, has been used to fight tuberculosis. Antibiotics are mainly useful for treating infections caused by bacteria. Antibiotics came into general use during the 1940’s. At the time, they were often called "wonder drugs" because they cured as many bacterial diseases that were once fatal. Some antibiotics are effective against infections caused by fungi and protozoa. Few antibiotics are useful in treating cancer. Antibiotics are also used to treat diseases in animals. The antibiotics support the animal’s growth for reasons that are not entirely understood. Farmers sometimes add small amounts of antibiotics to their livestock feed. This worries some scientists. Antibiotics are not effective against colds, influenza, or other viral diseases. The effectiveness of antibiotics is limited because both pathogenic microbes and cancer cells can become resistant to them.

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Effect of antibiotics on growth curve by using micro organisms

How Antibiotics Work Antibiotics fight microbes and cancer cells by interfering with normal cell functions. The interference occurs in one of three ways: prevention of cell wall formation, damage to cell membrane, or disruption of chemical processes. Dangers of Antibiotics A lot of antibiotics are regarded among the safest drugs when properly used. Antibiotics can sometimes cause dangerous side affects. The three main dangers are allergic reactions, destruction of helpful microbes, and damage to organs and tissues. Allergic Reactions In most cases allergic reactions are mild and produce only a rash of fever. Stern reactions can occur, and even cause death. All antibiotics are able to produce allergic reactions, but such reactions occur most often with penicillin’s. A physician usually asks if the patient has had any allergic reactions to any antibiotics before prescribing it

5.Bacterial mechanisms of antibiotic resistance :
Several mechanisms have evolved in bacteria which confer them with antibiotic resistance. These mechanisms can either chemically modify the antibiotic, render it inactive through physical removal from the cell, or modify target site so that it is not recognized by the antibiotic. The most common mode is enzymatic inactivation of the antibiotic. An existing cellular enzyme is modified to react with the antibiotic in such a way that it no longer affects the microorganism. An alternative strategy utilized by many bacteria is the alteration of the antibiotic target site. These and other mechanisms are shown in the the figure and accompanying table below.

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Mechanisms of antibiotic resistance in bacteria Antibiotic Chloramphenicol Tetracycline ?-lactams, Erythromycin, Lincomycin ?-lactams, Aminoglycosides, Chloramphenicol Sulfonamides, Trimethoprim Sulfonamides, Trimethoprim Method of resistance

reduced uptake into cell active efflux from the cell eliminates or reduces binding of antibiotic to cell target enzymatic cleavage or modification to inactivate antibiotic mol metabolic bypass of inhibited reaction overproduction of antibiotic target (titration)

The acquisition and spread of antibiotic resistance in bacteria: The development of resistance is inevitable following the introduction of a new antibiotic. Initial rates of resistance to new drugs are normally on the order of 1%. However, modern uses of antibiotics have caused a huge increase in the number of resistant bacteria. In fact, within 8-12 years after wide-spread use, strains resistant to multiple drugs become widespread. Multiple drug resistant strains of some bacteria have reached the proportion that virtually no antibiotics are available for treatment. Antibiotic resistance in bacteria may be an inherent trait of the organism (e.g. a particular type of cell wall structure) that renders it naturally resistant, or it may be acquired by means of mutation in its own DNA or acquisition of resistance-conferring DNA from another source. Inherent (natural) resistance: Bacteria may be inherently resistant to an antibiotic. For example, an organism lacks a transport system for an antibiotic; or an organism lacks the target of the antibiotic molecule; or, as in the case of Gram-negative bacteria, the cell wall is covered with an outer membrane

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that establishes a permeability barrier against the antibiotic. Acquired resistance: Several mechanisms are developed by bacteria in order to acquire resistance to antibiotics. All require either the modification of existing genetic material or the acquisition of new genetic material from another source. Vertical gene transfer : The spontaneous mutation frequency for antibiotic resistance is on the order of about of about 10-8- 10-9. This means that one in every every 108- 109 bacteria in an infection will develop resistance through the process of mutation. In E. coli, it has been estimated that streptomycin resistance is acquired at a rate of approximately 10-9 when exposed to high concentrations of streptomycin. Although mutation is a very rare event, the very fast growth rate of bacteria and the absolute number of cells attained means that it doesn't take long before resistance is developed in a population. Once the resistance genes have developed, they are transferred directly to all the bacteria's progeny during DNA replication. This is known as vertical gene transfer or vertical evolution. The process is strictly a matter of Darwinian evolution driven by principles of natural selection: a spontaneous mutation in the bacterial chromosome imparts resistance to a member of the bacterial population. In the selective environment of the antibiotic, the wild type (non mutants) are killed and the resistant mutant is allowed to grow and flourish Horizontal gene transfer : Another mechanism beyond spontaneous mutation is responsible for the acquisition of antibiotic resistance. Lateral or horizontal gene transfer (HGT) is a process whereby genetic material contained in small packets of DNA can be transferred between individual bacteria of the same species or even between different species. There are at least three possible mechanisms of HGT, equivalent to the three processes of genetic exchange in bacteria. These are transduction, transformation or conjugation. Conjugation occurs when there is direct cell-cell contact between two bacteria (which need not be closely related) and transfer of small pieces of DNA called plasmids takes place. This is thought to be the main mechanism of HGT. Transformation is a process where parts of DNA are taken up by the bacteria from the external environment. This DNA is normally present in the external environment due to the death and lysis of another bacterium. Transduction occurs when bacteria-specific viruses (bacteriophages) transfer DNA between two closely related bacteria.

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Mechanisms of horizontal gene transfer (HGT) in bacteria: The combined effects of fast growth rates to large densities of cells, genetic processes of mutation and selection, and the ability to exchange genes, account for the extraordinary rates of adaptation and evolution that can be observed in the bacteria. For these reasons bacterial adaptation (resistance) to the antibiotic environment seems to take place very rapidly in evolutionary time. Bacteria evolve fast!

Tests for sensitivity and resistance to antibiotics. (Left) The size of the zones of inhibition of microbial growth surrounding the antibiotic disks on the plate are an indication of microbial susceptibility to the antibiotic. (Right) By the use of these disks it is also possible to detect the occurrence of individual mutants within the culture that have developed antibiotic resistance. This image shows a close-up of the novobiocin disk (marked by an arrow on the whole plate) near which individual mutant cells in the bacterial population that were resistant to the antibiotic and have given rise to small colonies within the zone of inhibition

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Antibiotic sensitivity and resistance:
Ampicillin: is a penicillin derivative that inhibits crosslinking of peptidoglycan chains in the cell wall of eubacteria. Cells growing in the presence of ampicillin synthesize weak cell walls, causing them to burst due to the high internal osmotic pressure. AmpR encoded by Mu derivatives and pBR plasmids is due to a periplasmic ß-lactamase that breaks the ß-lactam ring of penicillin derivatives.

Tetracycline : inhibits protein synthesis. Tetracycline reversibly binds to the small subunit of ribosomes and interfere with binding of aminoacyl -tRNA to the Acceptor site. Tetracycline is bacteriostatic in bacteria. Tetracyclines can also inhibit protein synthesis in eukaryotes, but are less likely to reach inhibitory concentrations because eukaryotes lack a tetracycline uptake mechanism. TetR encoded by Tn10 and pBR plasmids is due to a membrane protein that actively exports tetracycline out of the cell. When Tn10 is present in multiple copies, cells are less resistant to Tet than when only one copy of Tn10 is present.

Spectinomycin : inhibits protein synthesis by binding to the Sr protein of the 30s ribosomal subunit and blocking translation. Spectinomycin resistance can arise via a single base substitution in the chromosomal gene encoding this ribosomal protein (rpsE). Plasmid encoded resistance to both spectinomycin and streptomycin can arise via modification of the antibiotics – for example, the aad gene transfers an adenyl group from ATP to these antibiotics which inactivates the antibiotic.

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Streptomycin inhibits protein synthesis by binding to the S12 protein of the 30s ribosomal subunit and blocking translation. A high level of Strr can result from chromosomal mutations in the gene for the S12 protein (rpsL) which prevent streptomycin from binding to the ribosome. Since only mutant ribosomes are StrR, resistance to streptomycin is recessive to streptomycin sensitivity. StrR requires phenotypic expression.

Antibiotics List Classification
In the following list are grouped characteristics of each family of antibiotics, indications, major adverse events, cons-indications. Aminoglycosides. Including inter alia streptomycin (used against tuberculosis) and gentamicin, are effective antibiotics against urinary tract infections and severe intestinal. Their toxicity is primarily auditory and renal (kidney and ear). Their main indications are cons-anesthesia, renal failure, pregnancy. The TB with ethambutol have liver toxicity, neurological and ocular (liver, nervous system and eyes). They are cons-indicated during pregnancy and in patients with impaired liver or kidney. Rifampicin and streptomycin are among TB. They are related drugs in the treatment of tuberculosis, but having a toxicity level of the ear, liver, kidneys, digestive system, and are also likely to cause allergic reactions. They are cons-indicated in cases of anesthesia, renal failure in infants, during pregnancy and in cases of allergy to these drugs. Betalactamines 1 containing penicillin G (penicillin V, penicillin F, a penicillin, ampicillin) are drugs used relatively common whose own indications are relatively large: Infections heart, skin, broncho-pulmonary, genital, ear, nose and throat, meningitis , digestive, bone, joint, urinary, Listeriosis, syphilis, etc. ... Their main side effects are possible allergic reactions, toxicity associated with neurological, renal and gastrointestinal. Their main indication is cons-allergy. In this class of antibiotics called carbapenems (imipenem) are reserved to the hospital for severe illnesses that are resistant to other antibiotics. The beta-lactam 2 include first generation cephalosporins with cefaclor, céfapirine, cefazolin. They have indicated anti-infectious very broad similar to penicillins. The second and third generations are reserved for hospital care and severe infections. The main side effects are allergic reactions associated with hemorrhage.

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The lincosanides. Including clindamycin part, are reserved for certain serious conditions, but have a digestive and liver toxicity. The main cons-lincosanides is an indication of liver failure. Macrolides. With erythromycin and josamycin are the common drugs indicated mainly in case of genital infection, ear, nose and throat, lung and for infringement by toxoplasmosis. The main side effects are allergic reactions and liver toxicity and gastrointestinal. The main cons-indication is liver failure. Nitroimidazoles. Including metronidazole, are shown in secondary infections with anaerobic bacteria, that is to say, can live without oxygen. The main side effect is gastrointestinal fragility. The main indication is cons-allergy medication. The phénocolés. Including the tiamphénicol, have indications in severe disease and failure of other antibiotics. However, their major side effects were gastrointestinal toxicity and blood. They are cons-indicated in pregnancy, infants and liver disease. The polypeptides. Which colistin belongs, have entries in the urinary infections. Their main side effects are toxic to the nervous system and kidneys. The main indications are cons-anesthesia and renal failure. Quinolones. With acid and nalidixic acid pipemidic are listed in the urinary and genital infections. Beware of allergic reactions. On the other hand, they have some toxicity hearing (inner ear). They are mainly cons-indicated in cases of epilepsy in some psychiatric illnesses

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during pregnancy and infant. The rifamycins. With rifamycin used mainly topical, may cause allergic reactions. Rifampicin is also part of this class of antibiotics, it is used against tuberculosis, but it has a toxic digestive and liver. On the other hand, it is cons-indicated in infants. Sulfonamides. Which may or not be associated with trimethoprim, which is part sulfadiazine and sulfamethoxazole, used in some congenital urinary tract infections but also in case of failure of other antibiotics. The main side effects and adverse reactions are allergies and toxicity in the blood and kidneys. Their main indications are cons-renal failure, pregnancy, infant. The synergistins. Including pristinamycin and virginiamycin are part, are shown in skin infections, lung and bone. Their toxicity is mainly gastrointestinal and liver. The main consindication is liver failure. Tetracyclines. With doxycycline, minocycline and tetracycline are common drugs primarily indicated in genital infections, cholera, typhus, lung ailments. The main side effects are allergic reactions, as well as neurological toxicity, renal and gastrointestinal. The main cons-indications are the children for eight years, severe liver or kidney. Various antibiotics. Including fusidic acid, vancomycin, fosfomycin and teicoplanin, are reserved to the hospital for staph infections and other severe infections. Their toxicity is in the inner ear and kidney. The main indications are cons-allergy and liver failure.

Types of Antibiotics
The different types of antibiotics are arranged according to their effective range in the antibiotics list.

Mainly four types of antibiotics : 5. 6. 7. 8. Ampcilline Penicillin Tetracycline Streptomycin

Ampicillin :

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Ampcilin chemical structure

Pharmacokinetic data
Bioavailability Protein binding Metabolism Half-life Excretion 40% (oral) 15 to 25% 12 to 50% approx 1 hour 75 to 85% renal

Identification :
CAS number ATC code PubChem DrugBank ChemSpider UNII KEGG ChEMBL 69-53-4 J01CA01 S01AA19 QJ51CA01 CID 6249 DB00415 6013 Y 7C782967RD Y D00204 N CHEMBL174 N

Chemical data
Formula Mol. mass C16H19N3O4S 349.41 g·mol?1

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SMILES

eMolecules & PubChem

Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. Until the introduction of ampicillin by the British company Beecham, penicillin therapies had only been effective against Gram-positive organisms such as staphylococci and streptococci. Ampicillin (originally branded as 'Penbritin') also demonstrated activity against Gram-negative organisms such as H. influenzae, coliforms and Proteus spp. Ampicillin was the first of a number of so-called broad spectrum penicillins subsequently introduced by Beecham. Ampicillin is part of the aminopenicillin family and is roughly equivalent to its successor, amoxicillin in terms of spectrum and level of activity. It can sometimes result in reactions that range in severity from a rash (in the case of patients that may unwittingly have mononucleosis) to potentially lethal allergic reactions such as anaphylaxis. However, as with other penicillin drugs, it is relatively non-toxic and adverse effects of a serious nature are encountered only rarely.

Mechanism of action
Belonging to the penicillin group of beta-lactam antibiotics, ampicillin is able to penetrate Grampositive and some Gram-negative bacteria. It differs from penicillin only by the presence of an amino group. That amino group helps the drug penetrate the outer membrane of gram-negative bacteria. Ampicillin acts as a competitive inhibitor of the enzyme transpeptidase, which is needed by bacteria to make their cell walls. It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, which ultimately leads to cell lysis. Ampicillin has received FDA approval for its mechanism of action.

Effects on chloroplast division
Ampicillin, like other ?-lactam antibiotics, not only blocks the division of bacteria, but also the division of chloroplasts of the Glaucophytes (called cyanelles) and chloroplasts of the moss Physcomitrella patens, a bryophyte. In contrast, it has no effect on the plastids of the higher developed vascular plant Lycopersicon esculentum L. (tomato).

Application
Ampicillin is closely related to amoxicillin, another type of penicillin, and both are used to treat urinary tract infections, otitis media, uncomplicated community-acquired pneumonia, Haemophilus influenzae, salmonellosis and Listeria meningitis. It is used with flucloxacillin in the combination antibiotic co-fluampicil for empiric treatment of cellulitis; providing cover against Group A streptococcal infection whilst the flucloxacillin acts against the Staphylococcus

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aureus bacterium. Of concern is the number of bacteria that become resistant to Ampicillin necessitating combination therapy or use of other antibiotics. All Pseudomonas and most strains of Klebsiella and Aerobacter are considered resistant. An ampicillin resistance gene (abbreviated bla) is commonly used as a selectable marker in routine biotechnology. Due to concerns over horizontal gene transfer to pathogenic organisms in the wild, the European Food Safety Authority restricts use of this gene (among other resistance genes) in commercial genetically modified organisms. The enzyme responsible for degrading ampicillin is called beta-lactamase, in reference to the beta-lactam structure of ampicillin and related drugs.

Penicillin :

Penicillin chemical structure
Penicillin core structure. The "R" is the variable group. Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from Penicillium fungi. They include penicillin G, procaine penicillin, benzathine penicillin, and penicillin V. Penicillin antibiotics are historically significant because they are the first drugs that were effective against many previously serious diseases such as syphilis and infections caused by staphylococci and streptococci. Penicillins are still widely used today, though many types of bacteria are now resistant. All penicillins are beta-lactam antibiotics and are used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms.

History
Discovery
Main article: History of penicillin

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The discovery of penicillin is attributed to Scottish scientist and Nobel laureate Alexander Fleming in 1928. He showed that, if Penicillium notatum were grown in the appropriate substrate, it would exude a substance with antibiotic properties, which he dubbed penicillin. This serendipitous observation began the modern era of antibiotic discovery. The development of penicillin for use as a medicine is attributed to the Australian Nobel laureate Howard Walter Florey together with the German Nobel laureate Ernst Chain and the English biochemist Norman Heatley. However, several others reported the bacteriostatic effects of Penicillium earlier than Fleming. The use of bread with a blue mould (it is presumed, penicillium) as a means of treating suppurating wounds was a staple of folk medicine in Europe since the middle Ages. The first published reference appears in the publication of the Royal Society in 1875, by John Tyndall.[16] Ernest Duchesne documented it in an 1897 paper, which was not accepted by the Institut Pasteur because of his youth. In March 2000, doctors at the San Juan de Dios Hospital in San José, Costa Rica, published the manuscripts of the Costa Rican scientist and medical doctor Clodomiro (Clorito) Picado Twight (1887–1944). They reported Picado's observations on the inhibitory actions of fungi of the genus Penicillium between 1915 and 1927. Picado reported his discovery to the Paris Academy of Sciences, yet did not patent it, even though his investigations started years before Fleming's. Joseph Lister was experimenting with penicillum in 1871 for his Aseptic surgery. He found that it weakened the microbes but then he dismissed the fungi. These early investigations did not lead to the use of antibiotics to treat infection because they took place in obscure circumstances, and the idea that infections were caused by transmissible agents was not widely accepted at the time. Sterilization measures had been shown to limit the outbreak and spread of disease; however, the mechanism of transmission of disease by parasites, bacteria, viruses and other agents was unknown. In the late 19th century, there was increasing knowledge of the mechanisms by which living organisms become infected, how they manage infection once it has begun and, most importantly in the case of penicillin, the effect that natural and man-made agents could have on the progress of infection. Fleming recounted that the date of his discovery of penicillin was on the morning of Friday, September 28, 1928. It was a fortuitous accident: in his laboratory in the basement of St. Mary's Hospital in London (now part of Imperial College), Fleming noticed a petri dish containing Staphylococcus plate culture he had mistakenly left open, which was contaminated by blue-green mould, which had formed a visible growth. There was a halo of inhibited bacterial growth around the mould. Fleming concluded that the mould was releasing a substance that was repressing the growth and lysing the bacteria. He grew a pure culture and discovered that it was a Penicillium mould, now known to be Penicillium notatum. Charles Thom, an American specialist working at the U.S. Department of Agriculture, was the acknowledged expert, and Fleming referred the matter to him. Fleming coined the term "penicillin" to describe the filtrate of a broth culture of the Penicillium mould. Even in these early stages, penicillin was found to be most effective against Gram-positive bacteria, and ineffective against Gram-negative organisms and fungi. He expressed initial optimism that penicillin would be a useful disinfectant, being highly potent with minimal toxicity compared to antiseptics of the day, and noted its laboratory

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value in the isolation of "Bacillus influenzae" (now Haemophilus influenzae).] After further experiments, Fleming was convinced that penicillin could not last long enough in the human body to kill pathogenic bacteria, and stopped studying it after 1931. He restarted clinical trials in 1934, and continued to try to get someone to purify it until 1940.

Production
Penicillin is a secondary metabolite of fungus Penicillium that is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin. ?-ketoglutarate + AcCoA ? homocitrate ? L-?-aminoadipic acid ? L-Lysine + ?lactam The by-product L-Lysine inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production. The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress, which is required for induction of penicillin production. The carbon sources that are available are also important: Glucose inhibits penicillin production, whereas lactose does not. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must also be carefully controlled. The biotechnology method of directed evolution has been applied to produce by mutation a large number of Penicillium strains. These techniques include error-prone PCR, DNA shuffling, ITCHY, and strand overlap PCR.

Developments from penicillin
The narrow range of treatable diseases or spectrum of activity of the penicillins, along with the poor activity of the orally active phenoxymethylpenicillin, led to the search for derivatives of penicillin that could treat a wider range of infections. The isolation of 6-APA, the nucleus of penicillin, allowed for the preparation of semisynthetic penicillins, with various improvements over benzylpenicillin (bioavailability, spectrum, stability, tolerance). The first major development was ampicillin, which offered a broader spectrum of activity than either of the original penicillins. Further development yielded beta-lactamase-resistant penicillins including flucloxacillin, dicloxacillin, and methicillin. These were significant for their activity against beta-lactamase-producing bacteria species, but are ineffective against the methicillinresistant Staphylococcus aureus strains that subsequently emerged. Another development of the line of true penicillins was the antipseudomonal penicillins, such as carbenicillin, ticarcillin, and piperacillin, useful for their activity against Gram-negative bacteria. However, the usefulness of the beta-lactam ring was such that related antibiotics, including the

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mecillinams, the carbapenems and, most important, the cephalosporins, still retain it at the center of their structures.

Mass production
The chemical structure of penicillin was determined by Dorothy Crowfoot Hodgkin in 1945. Penicillin has since become the most widely used antibiotic to date, and is still used for many Gram-positive bacterial infections. A team of Oxford research scientists led by Australian Howard Florey and including Ernst Boris Chain and Norman Heatley devised a method of massproducing the drug. Florey and Chain shared the 1945 Nobel Prize in Medicine with Fleming for their work. After World War II, Australia was the first country to make the drug available for civilian use. Chemist John C. Sheehan at MIT completed the first total synthesis of penicillin and some of its analogs in the early 1950s, but his methods were not efficient for mass production. The challenge of mass-producing this drug was daunting. On March 14, 1942, the first patient was treated for streptococcal septicemia with U.S.-made penicillin produced by Merck & Co. half of the total supply produced at the time was used on that one patient. By June 1942, there was just enough U.S. penicillin available to treat ten patientsIn July 1943, the War Production Board drew up a plan for the mass distribution of penicillin stocks to Allied troops fighting in Europe A moldy cantaloupe in a Peoria, Illinois, market in 1943 was found to contain the best and highest-quality penicillin after a worldwide search at the Northern Regional Research Laboratory at Peoria, Illinois, allowed the United States to produce 2.3 million doses in time for the invasion of Normandy in the spring of 1944. Large-scale production resulted from the development of deep-tank fermentation by chemical engineer Margaret Hutchinson RousseauAs a direct result of the war and the War Production Board, by June 1945 over 646 billion units per year were being produced. Penicillin was being mass-produced in 1944. G. Raymond Rettew made a significant contribution to the American war effort by his techniques to produce commercial quantities of penicillin. During World War II, penicillin made a major difference in the number of deaths and amputations caused by infected wounds among Allied forces, saving an estimated 12%–15% of lives. Availability was severely limited, however, by the difficulty of manufacturing large quantities of penicillin and by the rapid renal clearance of the drug, necessitating frequent dosing. Penicillin is actively excreted, and about 80% of a penicillin dose is cleared from the body within three to four hours of administration. Indeed, during the early penicillin era, the drug was so scarce and so highly valued that it became common to collect the urine from patients being treated, so that the penicillin in the urine could be isolated and reused. This was not a satisfactory solution, so researchers looked for a way to slow penicillin excretion. They hoped to find a molecule that could compete with penicillin for the organic acid transporter responsible for excretion, such that the transporter would preferentially excrete the competing molecule and the penicillin would be retained. The uricosuric agent probenecid proved to be suitable. When probenecid and penicillin are administered together, probenecid competitively inhibits the excretion of penicillin, increasing

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penicillin's concentration and prolonging its activity. Eventually, the advent of mass-production techniques and semi-synthetic penicillins resolved the supply issues, so this use of probenecid declined. Probenecid is still useful, however, for certain infections requiring particularly high concentrations of penicillins.

Medical uses
The term "penicillin" is often used generically to refer to benzylpenicillin (penicillin G), procaine benzyl penicillin (procaine penicillin), benzathine benzylpenicillin (benzathine penicillin), and phenoxymethylpenicillin (penicillin V). Procaine penicillin and benzathine penicillin have the same antibacterial activity as benzylpenicillin but act for a longer period. Phenoxymethylpenicillin is less active against Gramnegative bacteria than benzylpenicillin. Benzylpenicillin, procaine penicillin and benzathine penicillin are given by injection (parenterally), but phenoxymethylpenicillin is given orally. In 1930, Cecil George Paine, a pathologist at the Royal Infirmary in Sheffield, attempted to use penicillin to treat sycosis barbae, eruptions in beard follicles, but was unsuccessful, probably because the drug did not penetrate the skin deeply enough. Moving on to ophthalmia neonatorum, a gonococcal infection in infants, he achieved the first recorded cure with penicillin, on November 25, 1930. He then cured four additional patients (one adult and three infants) of eye infections, failing to cure a fifth. In 1939, Australian scientist Howard Florey (later Baron Florey) and a team of researchers (Ernst Boris Chain, Arthur Duncan Gardner, Norman Heatley, M. Jennings, J. Orr-Ewing and G. Sanders) at the Sir William Dunn School of Pathology, University of Oxford made significant progress in showing the in vivo bactericidal action of penicillin. Their attempts to treat humans failed because of insufficient volumes of penicillin (the first patient treated was Reserve Constable Albert Alexander), but they proved it harmless and effective on mice. Some of the pioneering trials of penicillin took place at the Radcliffe Infirmary in Oxford, England. These trials continue to be cited by some sources as the first cures using penicillin, though the Paine trials took place earlier On March 14, 1942, John Bumstead and Orvan Hess saved a dying patient's life using penicillin.

Adverse effects
Main article: Penicillin drug reaction Common adverse drug reactions (?1% of patients) associated with use of the penicillins include diarrhea, hypersensitivity, nausea, rash, neurotoxicity, urticaria, and superinfection (including candidiasis). Infrequent adverse effects (0.1–1% of patients) include fever, vomiting, erythema, dermatitis, angioedema, seizures (especially in epileptics), and pseudomembranous colitis

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Tetracycline:

Tetracycline Chemical structure

This article deals with the group of antibiotics known as the tetracyclines. For the specific antibiotic called ?tetracycline,? see tetracycline

The 4 rings of the basic tetracycline structure. Tetracyclines are a group of broad-spectrum antibiotics whose general usefulness has been reduced with the onset of bacterial resistance. Despite this, they remain the treatment of choice for some specific indications. They are so named for their four (?tetra-?) hydrocarbon rings (?-cycl-?) derivation (?-ine?). To be specific, they are defined as "a subclass of polyketides having an octahydrotetracene-2carboxamide skeleton".They are collectively known as "derivatives of polycyclic naphthacene carboxamide". Tetracyclines are generally used in the treatment of infections of the respiratory tract, sinuses, middle ear, urinary tract, and intestines, and is used in the treatment of gonorrhoea, especially in patients allergic to ?-lactams and macrolides; however, their use for these indications is less popular than it once was due to widespread resistance development in the causative organisms. Their most common current use is in the treatment of moderately severe acne and rosacea (tetracycline, oxytetracycline, doxycycline, or minocycline).

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Doxycycline is also used as a prophylactic treatment for infection by Bacillus anthracis (anthrax) and is effective against Yersinia pestis, the infectious agent of bubonic plague. It is also used for malaria treatment and prophylaxis, as well as treating elephantiasis. Tetracyclines remain the treatment of choice for infections caused by chlamydia (trachoma, psittacosis, salpingitis, urethritis, and L. venereum infection), Rickettsia (typhus, Rocky Mountain spotted fever), brucellosis, and spirochetal infections (borreliosis, syphilis, and Lyme disease). In addition, they may be used to treat anthrax, plague, tularemia, and Legionnaires' disease. They may have a role in reducing the duration and severity of cholera, although drug-resistance is occurring, and their effects on overall mortality is questioned. Demeclocycline has an additional use in the treatment of SIADH. Tetracycline derivatives are currently being investigated for the treatment of certain inflammatory disorders.

History
The first member of the group to be discovered is Chlortetracycline (Aureomycin) in the late 1940s by Dr. Benjamin Duggar, a scientist employed by Lederle Laboratories who derived the substance from a golden-colored, fungus-like, soil-dwelling bacterium named Streptomyces aureofaciens. Oxytetracycline (Terramycin) was discovered shortly afterwards by AC Finlay et al.; it came from a similar soil bacterium named Streptomyces rimosus. Robert Burns Woodward determined the structure of Oxytetracycline enabling Lloyd H. Conover to successfully produce tetracycline itself as a synthetic product. The development of many chemically altered antibiotics formed this group. In June 2005, tigecycline, the first member of a new subgroup of tetracyclines named glycylcyclines, was introduced to treat infections that are resistant to other antimicrobics including conventional tetracyclines. While tigecycline is the first tetracycline approved in over 20 years, other, newer versions of tetracyclines are currently in human clinical trials.

Administration
When ingested, it is usually recommended that the more water-soluble, short-acting tetracyclines (plain tetracycline, chlortetracycline, Oxytetracycline, demeclocycline and methacycline) be taken with a full glass of water, either two hours after eating, or two hours before eating. This is partly because most tetracyclines bind with food and also easily with magnesium, aluminium, iron, and calcium, which reduces their ability to be completely absorbed by the body. Dairy products, antacids, or preparations containing iron are particularly recommended to be avoided near the time of taking the drug. Partial exceptions to these rules occur for doxycycline and minocycline, which may be taken with food (though not iron, antacids, or calcium supplements).

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Minocycline, can be taken with dairy products because it does not chelate calcium as readily, although dairy products do decrease absorption of minocycline slightly.

Mechanism of action
Tetracycline antibiotics are protein synthesis inhibitors, inhibiting the binding of aminoacyltRNA to the mRNA-ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA translation complex. Tetracyclines also have been found to inhibit matrix metalloproteinases. This mechanism does not add to their antibiotic effects, but has led to extensive research on chemically modified tetracyclines or CMTs (like incyclinide) for the treatmet of rosacea, acne, and various types of neoplasms. Since incyclinide was announced to be ineffective for rosacea in September 2007, no drugs of this group will be marketed in the near-future.

Mechanism and resistance
Tetracycline inhibits cell growth by inhibiting translation. It binds to the 16S part of the 30S ribosomal subunit and prevents the amino-acyl tRNA from binding to the A site of the ribosome. The binding is reversible in nature. Cells become resistant to tetracycline by at least three mechanisms: enzymatic inactivation of tetracycline, efflux, and ribosomal protection. Inactivation is the rarest type of resistance, where an acetyl group is added to the molecule, causing inactivation of the drug. In efflux, a resistance gene encodes a membrane protein that actively pumps tetracycline out of the cell. This is the mechanism of action of the tetracycline resistance gene on the artificial plasmid pBR322. In ribosomal protection, a resistance gene encodes a protein that can have several effects, depending on what gene is transferred. Six classes of ribosomal protection genes/proteins have been found, all with high sequence homology, suggesting a common evolutionary ancestor. Possible mechanisms of action of these protective proteins include: 1. blocking tetracyclines from binding to the ribosome 2. binding to the ribosome and distorting the structure to still allow t-RNA binding while tetracycline is bound 3. Binding to the ribosome and dislodging tetracycline. All of these changes to ribosomes are reversible (non-covalent) because ribosomes isolated from both tetracycline-resistant and susceptible organisms bind tetracycline equally well in vitro.

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Side effects
Side-effects from tetracyclines are not always common, but of particular note is photo toxicity. It increases the risk of sunburn under exposure to light from the sun or other sources. This may be of particular importance for those intending to take on vacations long-term doxycycline as a malaria prophylaxis. They may cause stomach or bowel upsets, and, on rarely occasions, allergic reactions. Very rarely, severe headache and vision problems may be signs of dangerous secondary intracranial hypertension, also known as pseudotumor cerebra. Tetracyclines are teratogens due to the likelihood of causing teeth discolouration in the fetus as they develop in infancy. For this same reason, tetracyclines are contraindicated for use in children under 8 years of age. They are, however, safe to use in the first 18 weeks of pregnancy. Some patients taking tetracyclines require medical supervision because they can cause steatosis and hepatotoxicity.

Cautions
Tetracyclines should be used with caution in those with liver impairment and those that are soluble in water and urine worsen renal failure (this is not true of the lipid soluble agent’s doxycycline and minocycline). They may increase muscle weakness in myasthenia gravis and exacerbate systemic lupus erythematosus. Antacids reduce the absorption of all tetracyclines, and dairy products reduce absorption greatly for all but minocycline. The breakdown products of tetracyclines are toxic and can cause Fanconi Syndrome, a potentially fatal disease affecting proximal tubular function in the nephrons of the kidney. Prescriptions of these drugs should be discarded once expired because they can cause hepatotoxicity. It was once believed that tetracycline antibiotics impair the effectiveness of many types of hormonal contraception. Recent research has shown no significant loss of effectiveness in oral contraceptives while using most tetracyclines. Despite these studies, many physicians still recommend the use of barrier contraception for people taking any tetracyclines to prevent unwanted pregnancy.

Contraindications
Tetracycline use should be avoided in pregnant or lactating women, and in children with developing teeth because they may result in permanent staining (dark yellow-gray teeth with a darker horizontal band that goes across the top and bottom rows of teeth), and possibly affect the growth of teeth and bones.

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In tetracycline preparation, stability must be considered in order to avoid formation of toxic epianhydrotetracyclines

Examples
According to source:
?

?

Naturally occurring o Tetracycline o Chlortetracycline o Oxytetracycline o Demeclocycline Semi-synthetic o Doxycycline o Lymecycline o Meclocycline o Methacycline o Minocycline o Rolitetracycline

According to duration of action:
?

?

?

Short-acting (Half-life is 6-8 hrs) o Tetracycline o Chlortetracycline o Oxytetracycline Intermediate-acting (Half-life is ~12 hrs) o Demeclocycline o Methacycline Long-acting (Half-life is 16 hrs or more) o Doxycycline o Minocycline o Tigecycline

Tigecycline may also be considered a tetracycline antibiotic, though it is usually classified as a glycylcycline antibiotic.

Streptomycin:

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Streptomycin chemical structure

Pharmacokinetic data
Bioavailability Half-life Excretion 84% to 88% (est.)[2] 5 to 6 hours Renal

Identification

CAS number ATC code PubChem DrugBank ChemSpider UNII KEGG ChEMBL

57-92-1 A07AA04 J01GA01 CID 19649 DB01082 18508 Y45QSO73OB D08531 CHEMBL1201194

Chemical data

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Formula Mol. mass SMILES

C21H39N7O12 581.574 g/mol eMolecules & PubChem

Physical data

Melt. point

12 °C (54 °F)

Streptomycin is an antibiotic drug, the first of a class of drugs called aminoglycosides to be discovered, and was the first antibiotic remedy for tuberculosis. It is derived from the actinobacterium Streptomyces griseus. Streptomycin is a bactericidal antibiotic. Streptomycin cannot be given orally, but must be administered by regular intramuscular injections. An adverse effect of this medicine is ototoxicity. Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This leads to codon misreading, eventual inhibition of protein synthesis and ultimately death of microbial cells through mechanisms that are still not understood. Humans have structurally different ribosomes from bacteria, thereby allowing the selectivity of this antibiotic for bacteria. However at low concentrations Streptomycin only inhibits growth of the bacteria by inducing prokaryotic ribosomes to misread mRNA. Streptomycin is an antibiotic that inhibits both Gram-positive and Gram-negative bacteria, and is a therefore a useful broad-spectrum antibiotic. Streptomycin was first isolated on October 19, 1943 by Albert Schatz, a graduate student, in the laboratory of Selman Abraham Waksman at Rutgers University. Waksman and his laboratory discovered several antibiotics, including actinomycin, clavacin, streptothricin, streptomycin, grisein, neomycin, fradicin, candicidin and candidin. Of these, streptomycin and neomycin found extensive application in the treatment of numerous infectious diseases. Streptomycin was the first antibiotic that could be used to cure the disease tuberculosis; early production of the drug was dominated by Merck & Co. under George W. Merck. The first randomized trial of streptomycin against pulmonary tuberculosis was carried out in 1946-1947 by the MRC Tuberculosis Research Unit under the chairmanship of Sir Geoffrey Marshall (1887–1982). The trial was both double-blind and placebo-controlled. It is widely accepted to have been the first randomised curative trial. Results showed efficacy against TB, albeit with minor toxicity and acquired bacterial resistance to the drug.

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Uses
Treatment of diseases
? ?

? ?

Infective endocarditis caused by enterococcus when the organism is not sensitive to Gentamicin Tuberculosis in combination with other anti-TB drugs. It is not the first-line treatment, except in medically under-served populations where the cost of more expensive treatments are prohibitive. Plague (Yesinia pestis) has historically been treated with it as the first-line treatment. It is approved for this purpose by the U.S. Food and Drug Administration. In veterinary medicine, streptomycin is the first-line antibiotic for use against gram negative bacteria in large animals (horses, cattle, sheep etc.). It is commonly combined with procaine penicillin for intramuscular injection.

While streptomycin is traditionally given intramuscularly (indeed, in many countries it is only licensed to be used intramuscularly), the drug may also be administered intravenously.

Pesticide
Streptomycin is also used as a pesticide, to combat the growth of bacteria, fungi, and algae. Streptomycin controls bacterial and fungal diseases of certain fruit, vegetables, seed, and ornamental crops, and controls algae in ornamental ponds and aquaria. A major use is in the control of fireblight on apple and pear trees. As in medical applications, extensive use can be associated with the development of resistant strains. Cell culture Streptomycin, in combination with penicillin, is used in a standard antibiotic cocktail to prevent bacterial infection in cell culture.

7. Materials Using effect of Antibiotics:
1. Autoclave 2.Laminarair flow 3.Incubator 4. Nephelometer 5. UV Spetrophotometer

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8.1 Autoclave:
In preparing items for autoclaving, containers should be unsealed and articles should be wrapped in materials that allow steam penetration. Large packages of dressings and large flasks of media require extra time for heat to penetrate them. Likewise, packing many articles close together in an autoclave lengthens the processing time to as much as 60 minutes to ensure sterility. It is more efficient and safer to run two separate, uncrowded loads than one crowded one. Wrapping objects in aluminum foil is not recommended because it may interfere with steam penetration. Steam circulates through an autoclave from a steam outlet to an air evacuation port

Importance: Moist heat in the form of pressurized steam is regarded as the most dependable method for the destruction of all forms of life, including bacterial spores. This method is incorporated into a device called the autoclave. Over 100 years ago, French and German microbiologist developed the autoclave as an essential component of their laboratories. Need of autoclaving: Reliable sterilization with moist heat requires temperatures above that of boiling water. These high temperatures are most commonly achieved by steam under pressure in an autoclave. Autoclaving is the preferred method of sterilization, unless the material to be sterilized can be damaged by heat or moisture. Effectiveness of Autoclave or Optimum Conditions: Sterilization in an autoclave is most effective when the organisms are either contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure about 15 psi; attaining temperature (121oC) will kill all organisms and their endospores in about 15 minutes.

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Principle of Autoclaving: A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally. For example, when free flowing steam at a temperature of 100oC is placed under a pressure of 1 atmosphere above sea level pressure – that is, about 15 pounds of pressure per square inch (Psi) --- the temperature rises to 121oC. Increasing the pressure to 20 psi raises the temperature to 126oC. The relationship between temperature and pressure is shown in table 2. In this way steam is a gas, increasing its pressure in a closed system increases its temperature. As the water molecules in steam become more energized, their penetration increases substantially. This principle is used to reduce cooking time in the home pressure cooker and to reduce sterilizing time in the autoclave. It is important to note that the sterilizing agent is the moist heat, not the pressure. Table Pressure (psi in excess of atmospheric pressure) 0 psi 5 psi 10 psi 15 psi 20 psi 30 psi Rules implied for Autoclaving: Sterilization by autoclaving is invariably successful if properly done and if two common-sense rules are followed: First, articles should be placed in the autoclave so that steam can easily penetrate them. Second, air should be evacuated so that the chamber fills with steam. Working of Autoclave: Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket where steam is maintained. As steam flows from the steam jacket into the sterilizing chamber, cool air is forced out and a special valve increases the pressure to 15 pounds/square inch above normal atmospheric pressure. The temperature rises to 121.5oC, and the superheated water molecules rapidly conduct heat into microorganisms. The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes. For denser objects, up to 30 minutes of exposure may be required. The conditions must be carefully controlled or serious problems may occur. Uses of Autoclave: Autoclaving is used to sterilize culture media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items that can withstand high temperatures and pressures. The laboratory technician uses it to sterilize bacteriological media and The Relationship Between the Pressure and Temperature of Steam at Sea Level* Temperature (oC) 100 110 116 121 126 135

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destroy pathogenic cultures. The autoclave is equally valuable for glassware and metalware, and is among the first instruments ordered when a microbiology laboratory is established. Autoclaves are also used on large industrial scale. Large industrial autoclaves are called retorts, but the same principle applies for common household pressure cooker used in the home canning of foods Limitations and Disadvantages of Autoclave: The autoclave also has certain limitations. For example, some plasticware melts in the high heat, and sharp instruments often become dull. Moreover, many chemicals breakdown during the sterilization process and oily substances cannot be treated because they do not mix with water. Heat requires extra time to reach the center of solid materials, such as caned meats, because such materials do not develop the efficient heat-distributing convection currents that occur in liquids. Heating large containers also requires extra time. Table 3 shows the different time requirements for sterilizing liquids in various container sizes. Unlike sterilizing aqueous solutions, sterilizing the surface of a solid requires that steam actually contact it. Table 3 Container Size Test Tube: 18×150 mm Erlenmeyer Flask: 95 ml 125 ml Erlenmeyer Flask:2000 ml 1500 ml Fermentation Bottle: 6750 ml 9000 ml 15 30 70 The effect of Container Size on Autoclve Sterilization Times for Liquid Solutions* Liquid Volume 10 ml

Sterilization Time (min) 15

Indicator of Sterilization Achievement: Several commercially available methods can indicate whether sterilization has been achieved by heat treatment. Modern autoclaves have devices to maintain proper pressure and record internal temperature during operations. Regardless of the presence of such a device, the operator should check pressure periodically and maintain the appropriate pressure. Chemical reactions in which an indicator changes color when the proper times and temperatures have been reached. In some designs, the word "sterile" or "autoclaved" appears on wrappings or tapes. These tapes are not fully reliable because they do not indicate how long appropriate conditions were maintained. Tapes or other sterilization indicators should be placed inside and near the center of large packages of determine whether heat penetrated them. In another method, a pellet contained within a glass vial melts. A widely used test consists of preparations of specified species of bacterial endospores such as Bacillus stearothermophilus, impregnated into paper strips. The spore strip and an ampule of medium are enclosed in a soft plastic vial. The vial is placed in the center of the material to be sterilized and is autoclaved. After autoclaving, these can then be

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aseptically inoculated into culture media. Growth in the culture media indicates survival of the endospores and therefore inadequate processing. Other designs use endospore suspensions that can be released, after heating, into a surrounding culture medium within medium within the same vial. Important Points to Remember For Autoclaving: Steam under pressure fails to sterilize when the air is not completely exhausted. This can happen with the premature closing of autoclave's automatic ejector valve. The principles of heat sterilization have a direct bearing on home canning. To sterilize dry glassware, bandages, and the like, care must be taken to ensure that steam contacts all surfaces. For example, aluminum foil is impervious to steam and should not be used to wrap dry materials that are to be sterilized; paper should be used instead. Care should also be taken to avoid trapping air In the bottom of a dry container because trapped air will not be replaced by steam, which is lighter than air. The trapped air is the equivalent of a small hot-air oven, which, as we will see shortly, requires a higher temperature and longer time to sterilize materials. Containers that can trap air should be placed in a tipped position so that the steam will force out the air. Products that do not permit penetration by moisture, such as mineral oil or petroleum jelly, are not sterilized by the same methods that would sterilize aqueous solutions. This precaution is necessary because when an object is exposed to heat, its surface becomes hot much more quickly than its center. (When a large piece of meat is roasted, for example, the surface can be well done while the center remains rare.) Prevacuum Autoclave: In large laboratories and hospitals, where great quantities of materials must be sterilized, special autoclaves, called prevacuum autoclaves, are often used. This machine draws air out of the sterilizing chamber at the beginning of the cycle. Saturated steam is then used at a temperature of 132oC to 134oC at a pressure of 28 to 30 lb/in2. The time for sterilization is now reduced to as little as 4 minutes. A vacuum pump operates at the end of the cycle to remove the steam and dry the load. The major advantages of the prevacuum autoclave are the minimal exposure time for sterilization, the reduced time to complete the cycle and the costs of sterilization are greatly decreased.

8.2Working for laminar air flow:
Safety: The finished product should be free of contamination (particles, bacteria, extraneous material). b.) The solution should be clear -- all medications should be completely dissolved. c.) All compounding materials should be checked for expiration date, outer integrity, etc. Accuracy: Guidelines must be set up to ensure the right drug, right dose, and right concentration. This includes using the appropriate syringe size to measure out fluid volumes in order to minimize errors. Another example would be to require that all syringes be drawn back to the original amount of each individual dose and placed next to the admixture to facilitate checking by the pharmacist. If a filter needle was required, it should also be present.

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Dangers of poor aseptic technique: Patients who are receiving intravenous therapy tend to be the most critical. Every precaution must be taken to avoid contamination. The IV route is the most dangerous route of administration because it bypasses all of the body's natural barriers. An improperly prepared solution when administered can have very serious consequences: infections, emboli, occlusions and even..

Laminar Flow Hoods:
1. Provide clean air to the working area.

2. Provide a constant flow of air out of the work area to prevent room air from entering. 3. The air flowing out from the hood suspends and removes contaminants introduced into the work area by personnel

The most important part of a laminar flow hood is a high efficiency bacteriaretentive filter. Room air is taken into the unit and passed through a pre-filter to remove gross contaminants (lint, dust etc). The air is then compressed and channeled up behind and through the HEPA filter (High Efficiency Particulate Air filter) in a laminar flow fashion--that is the purified air flows out over the entire work surface in parallel lines at a uniform velocity. The HEPA filter removes nearly all of the bacteria from the air. Why control room air? The environmental control of air is of concern because room air may be highly contaminated. Example: Sneezing produces 100,000 - 200,000 aerosol droplets which can then attach to dust particles. These contaminated particles may be present in the air for weeks. (Have you ever viewed the air around you when you open the curtains on a sunny day?)... Limitations: With poor technique it is easy to overcome the established airflow

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Effect of antibiotics on growth curve by using micro organisms

velocity and introduce reverse currents that can re-introduce contaminants into the work area. Laminar hoods should remain on 24 hours a day. If turned off for any reason, it should be on for at least 30 minutes and thoroughly cleaned before reusing.

Examples of vertical and horizontal laminar flow hoods:

Aseptic technique: guidelines & key points:
A direct path must be maintained between the filter and the area inside the hood where the manipulations are being performed. Air downstream from non-sterile objects (such as solution containers, hands etc.) becomes contaminated from particles blown off these objects. To best illustrate this very important point click on the two examples of proper aseptic technique: (manipulation of a vial and an ampoule)

1. Always minimize clutter. Waste and other items should never enter the hood. All calculations should be done before entering the hoodWash hands and arms

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Effect of antibiotics on growth curve by using micro organisms

2.

3.

4.

5. 6.

7.

before compounding or re-entering the hood. Also, remove any jewelry from the hands and wrists. It is important that you keep your hands within the cleaned area of the hood as much as possible. Do not touch your hair, face or clothing. Excess dust should be removed from items before introducing them into the hood When working in a horizontal laminar flow hood, all work must be performed at a distance of no less than 6 inches from the front edge of the work surface. At a distance of less than 6 inches, laminar flow air begins to mix with the outside air and contamination is possible. Never become so engrossed in your work that you forget this basic rule. Outer pouches and wraps should be removed at the edge of the work area as the sterile contents are pulled into the work area. Never bring these items into the main work area. Large objects should never be placed near the back of the hood. Not only do these objects contaminate everything downstream, but they also disrupt the laminar flow pattern of air which normally suspends the contaminants and removes them from the area. Remember that hand cleanliness is further reduced each time more bottles and other non-sterile items are handled. Before and after preparing a series of IV admixtures, or anytime something is spilled, the work surface of the laminar flow hood should be thoroughly cleaned with alcohol. A long side to side motion should be used starting at the back of the hood and then working forward. The acrylic plastic sides should also be cleaned periodically. It is possible to overcome the established airflow velocity by a strong reverse current produced by coughing, quick movements, talking etc. Keep all of these to a minimum in order to maintain a sterile environment. Do not talk, cough or sneeze into the hood!

8. The contents of glass ampoules should always be filtered before adding to an IV admixture.

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8.3Lab Incubator:
We are engaged in the sphere of fabricating a wide range of lab incubator that are available in different sizes and can also be customized as per the specifications, detailed by our clients. These are double walled and its door has double viewing window to offer super functionality. Specifications :
? ? ? ? ? ? ? ?

Double Walled, Inner Chamber fabricated with SS 304 Outer made of MS duly Powder coated. Insulated door has double viewing window 75mm gap between outer & inner wall is insulated with high efficiency mineral wool Temperature Range: Ambient to 70°C Temperature controlled by thermostat / Digital Temperature Indicator cum Controller having an accuracy of ± 0.5 °C Power supply: 220/230 V AC, Single phase

Suitable for safe treatment of microbiological cultures. The INCUCELL line produces no noise and provides a very soft air convection within the chamber. These devices can be used especially in biological and microbiological laboratories, duality tests in pharmacy, cosmetics and testing in veterinary medicine and food processing industry.

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Effect of antibiotics on growth curve by using micro organisms

? ? ? ?

Internal dim. of the chamber: 22, 55, 111, 222, 404, 707 litres Working temperature: from 5 °C above ambient temperature ap to 70 °C/99,9 °C Inner glas door Interior: stainles steel, mat No.1.4301 (AISI 304)

8.4 Working For Nephelometer :
A nephelometer [1] is a stationary or portable instrument for measuring suspended particulates in a liquid or gas colloid. A nephelometer measures suspended particulates by employing a light beam (source beam) and a light detector set to one side (often 90°) of the source beam. Particle density is then a function of the light reflected into the detector from the particles. To some extent, how much light reflects for a given density of particles is dependent upon properties of the particles such as their shape, color, and reflectivity. Nephelometers are calibrated to a known particulate, commonly Arizona road dust then use environmental factors k-factors to compensate lighter or darker colored dusts accordingly. K-factor is determined by the user by running the nephelometer next to an air sampling pump and comparing results. The Nephelometer is an analytical instrument used to measure the light-scattering coefficient of atmospheric and indoor aerosols. It measures the particulates in the air. The main uses of Nephelometers relate to air quality measurement for pollution monitoring, climate monitoring and visibility.

Particulate Contaminants:
This chart shows the types and sizes of various particulate contaminants. This information is helpful toward understanding the character of particulate pollution inside a building or in the ambient air. It is also useful for understanding the cleanliness level in a controlled environment.

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Biological contaminants include mold, fungus, bacteria, viruses, animal dander, dust mites, pollen, human skin cells, cockroach parts, or anything alive or living at one time. These are the biggest enemy of Indoor Air Quality specialist because they are contaminants that cause health problems. Levels of biological contamination depend on humidity and temperature that supports the livelihood of micro-organisms. The presence of pets, plants, rodents, and insects will raise the level of biological contamination

Visibility:
Nephelometers are also used for measurement of visibility with simple one-wavelength nephelometers used throughout the world by many EPAs. Nephelometers, through the measurement of light scattering, can determine visibility in distance through the application of a conversion factor called Koschmieder’s formula.
?

?

?

? ? ? ? ? ? ? ? ? ? ?

Because optical properties depend on suspended particle size, a stable synthetic material called "Formazin" with uniform particle size is often used as a standard for calibration and reproducibility.[2] The unit is called Formazin Turbidity Unit (FTU). Nephelometric Turbidity Units (NTU) specified by United States Environmental Protection Agency is a special case of FTU, where a white light source and certain geometrical properties of the measurement apparatus are specified. (Sometimes the alternate form "nephelos turbidity units" is used[3][4]) Formazin Nephelometric Units (FNU), prescribed for 9 measurements of turbidity in water treatment by ISO 7027, another special case of FTU with near infrared light (NIR) and 90° scatter. Formazin Attenuation Units (FAU) specified by ISO 7027 for water treatment standards for turbidity measurements at 0°, also a special case of FTU. Formazin Backscatter Units (FBU), not part of a standard, is the unit of optical backscatter detectors (OBS), measured at c. 180°, also a special case of FTU. European Brewery Convention (EBC) turbidity units Concentration Units (C.U.) Optical Density (O.D.) Jackson "Candle" Turbidity Units (JTU; an early measure) Helms Units American Society of Brewing Chemists (ASBC-FTU) turbidity units Parts Per Million of standard substance, such as PPM/DE (Kieselguhr) "Trübungseinheit/Formazin" (TE/F) a German standard, now replaced by the FNU unit. diatomaceous earth ("ppm SiO2") an older standard, now obsolete

A more popular term for this instrument in water quality testing is a turbidimeter. However, there can be differences between models of turbidimeters, depending upon the arrangement (geometry) of the source beam and the detector. A nephelometric turbidimeter always monitors light reflected off the particles and not attenuation due to cloudiness. In the United States environmental monitoring the turbidity standard unit is called Nephelometric Turbidity Units (NTU), while the international standard unit is called Formazin Nephelometric Unit (FNU). The

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most generally applicable unit is Formazin Turbidity Unit (FTU), although different measurement methods can give quite different values as reported in FTU (see below). Gas-phase nephelometers are also used to study the atmosphere. These can provide information on visibility and atmospheric albedo. Gas-phase nephelometers are also used in the detection of smoke & other particles of combustion. In such use, the apparatus is referred to as an aspirated smoke detector. These have the capability to detect extremely low particle concentrations (to 0.005%) and are therefore highly suitable to protecting sensitive or valuable electronic equipment, such as mainframe computers and telephone switches.

Nephelometer uses:
The main uses of nephelometers relate to air quality measurement for pollution monitoring, climate monitoring, and visibility. Airborne particles are commonly either biological contaminants, particulate contaminants, gaseous contaminants, or dust.

8.5 Visible and Ultraviolet Spectroscopy:
An obvious difference between certain compounds is their color. Thus, quinone is yellow; chlorophyll is green; the 2,4-dinitrophenylhydrazone derivatives of aldehydes and ketones range in color from bright yellow to deep red, depending on double bond conjugation; and aspirin is colorless. In this respect the human eye is functioning as a spectrometer analyzing the light reflected from the surface of a solid or passing through a liquid. Although we see sunlight (or white light) as uniform or homogeneous in color, it is actually composed of a broad range of radiation wavelengths in the ultraviolet (UV), visible and infrared (IR) portions of the spectrum. As shown on the right, the component colors of the visible portion can be separated by passing sunlight through a prism, which acts to bend the light in differing degrees according to wavelength. Electromagnetic radiation such as visible light is commonly treated as a wave phenomenon, characterized by a wavelength or frequency. Wavelength is defined on the left below, as the distance between adjacent peaks (or troughs), and may be designated in meters, centimeters or nanometers (10-9 meters). Frequency is the number of wave cycles that travel past a fixed point per unit of time, and is usually given in cycles per second, or hertz (Hz). Visible wavelengths cover a range from approximately 400 to 800 nm. The longest visible wavelength is red and the shortest is violet. Other common colors of the spectrum, in order of decreasing wavelength, may be remembered by the mnemonic: ROY G BIV. The wavelengths of what we perceive as particular colors in the visible portion of the spectrum are displayed and listed below. In horizontal diagrams, such as the one on the bottom left, wavelength will increase on moving from left to right.

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? ? ? ? ? ? ?

Violet: Indigo: Blue: Green: Yellow: Orange: Red:

400 - 420 nm 420 - 440 nm 440 - 490 nm 490 - 570 nm 570 - 585 nm 585 - 620 nm 620 - 780 nm

When white light passes through or is reflected by a colored substance, a characteristic portion of the mixed wavelengths is absorbed. The remaining light will then assume the complementary color to the wavelength(s) absorbed. This relationship is demonstrated by the color wheel shown on the right. Here, complementary colors are diametrically opposite each other. Thus, absorption of 420-430 nm light renders a substance yellow, and absorption of 500520 nm light makes it red. Green is unique in that it can be created by absoption close to 400 nm as well as absorption near 800 nm. Early humans valued colored pigments, and used them for decorative purposes. Many of these were inorganic minerals, but several important organic dyes were also known. These included the crimson pigment, kermesic acid, the blue dye, indigo, and the yellow saffron pigment, crocetin. A rare dibromo-indigo derivative, punicin, was used to color the robes of the royal and wealthy. The deep orange hydrocarbon carotene is widely distributed in plants, but is not sufficiently stable to be used as permanent pigment, other than for food coloring. A common feature of all these colored compounds, displayed below, is a system of extensively conjugated pi-electrons.

2. The Electromagnetic Spectrum:

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The visible spectrum constitutes but a small part of the total radiation spectrum. Most of the radiation that surrounds us cannot be seen, but can be detected by dedicated sensing instruments. This electromagnetic spectrum ranges from very short wavelengths (including gamma and xrays) to very long wavelengths (including microwaves and broadcast radio waves). The following chart displays many of the important regions of this spectrum, and demonstrates theinverse relationship between wavelength and frequency The energy associated with a given segment of the spectrum is proportional to its frequency. The bottom equation describes this relationship, which provides the energy carried by a photon of a given wavelength of radiation To obtain specific frequency, wavelength and energy values use this calculator. 3. UV-Visible Absorption Spectra: To understand why some compounds are colored and others are not, and to determine the relationship of conjugation to color, we must make accurate measurements of light absorption at different wavelengths in and near the visible part of the spectrum. Commercial optical spectrometers enable such experiments to be conducted with ease, and usually survey both the near ultraviolet and visible portions of the spectrum.The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mole, and the near ultraviolet region, out to 200 nm, extends this energy range to 143 kcal/mole. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle, and is seldom used as a routine tool for structural analysis.The corrected absorption value is called "molar absorptivity", and is particularly useful when comparing the spectra of different compounds and determining the relative strength of light absorbing functions (chromophores). Molar absorptivity (?) is defined as: Molar Absorptivity, ? = A /cl (where A= absorbance, c = sample concentration in moles/liter & l = length of light path through the sample in cm.)

If the isoprene spectrum on the right was obtained from a dilute hexane solution (c = 4 * 10-5 moles per liter) in a 1 cm sample cuvette, a simple calculation using the above formula indicates a molar absorptivity of 20,000 at the maximum absorption wavelength. Indeed the entire vertical absorbance scale may be changed to a molar absorptivity scale once this information about the sample is in hand. Clicking on the spectrum will display this change in units.

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Chromophore C=C C?C C=O N=O C-X X=Br X=I

Example Ethene 1-Hexyne Ethanal Nitromethane Methyl bromide Methyl Iodide

Excitation ? ? n ? n ? n n
__ __

?max, nm 171 180 290 180 275 200 205 255

? 15,000 10,000 15 10,000 17 5,000 200 360

Solvent hexane hexane hexane hexane ethanol ethanol hexane hexane

> ?* > ?* > ?* > ?* > ?* > ?*

__

__ __

__ __ __

> ?* > ?*

4. The Importance of Conjugation: A comparison of the absorption spectrum of 1-pentene, ?max = 178 nm, with that of isoprene (above) clearly demonstrates the importance of chromophore conjugation. Further evidence of this effect is shown below. The spectrum on the left illustrates that conjugation of double and triple bonds also shifts the absorption maximum to longer wavelengths. From the polyene spectra displayed in the center diagram, it is clear that each additional double bond in the conjugated pielectron system shifts the absorption maximum about 30 nm in the same direction. Also, the molar absorptivity (?) roughly doubles with each new conjugated double bond. Spectroscopists use the terms defined in the table on the right when describing shifts in absorption. Thus, extending conjugation generally results in bathochromic and hyperchromic shifts in absorption. The appearance of several absorption peaks or shoulders for a given chromophore is common for highly conjugated systems, and is often solvent dependent. This fine structure reflects not only the different conformations such systems may assume, but also electronic transitions between the different vibrational energy levels possible for each electronic state. Vibrational fine structure of this kind is most pronounced in vapor phase spectra, and is increasingly broadened and obscured in solution as the solvent is changed from hexane to methanol.

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9.1PREPARATION OF NUTRIENT AGAR:
Bacteriological media come an a wide range of types. Nutrient Agar is a complex medium because it contains ingredients with contain unknown amounts or types of nutrients. Nutrient Agar contains Beef Extract (0.3%), Peptone (0.5%) and Agar (1.5%) in water. Beef extract is the commercially prepared dehydrated form of autolysed beef and is supplied in the form of a paste. Peptone is casein (milk protein) that has been digested with the enzyme pepsin. Peptone is dehydrated and supplied as a powder. Peptone and Beef Extract contain a mixture of amino acids and peptides. Beef Extract also contains water soluble digest products of all other macromolecules (nucleic acids, fats, polysaccharides) as well as vitamins and trace minerals. Although we know and can define Beef Extract in these terms, each bach can not be chemically defined. There are many media ingredients which are complex: yeast extract, tryptone, and others. The advantage of complex media is that they support the growth of a wide range of microbes.

Agar is purified from red algae in which it is an accessory polysaccharide (polygalacturonic acid) of their cell walls. Agar is added to microbiological media only as a solidification agent. Agar for most purposes has no nutrient value. Agar is an excellent solidification agent because it dissolves at near boiling but solidifies at 45oC. Thus, one can prepare molten (liquid) agar at 45oC, mix cells with it, then allow it to solidify thereby trapping living cells. Below 45oC agar is a solid and remains so as the temperature is raised melting only when >95oC is obtained.

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MATERIALS: 1. Electronic or beam balances. 2. Weigh boats, tongue depressors. 3. Tripods, asbestos wire-gauze, asbestos gloves. 4. 10 ml nonsterile pipettes. 5. pH paper or pH meter with standard buffers. 6. 4 13x100 mm screw capped culture tubes. 7. Graduated Cylinder, 250 ml. 8. 2 500ml Erlenmeyer Flasks 9. Beef Extract, Peptone, Agar. 10. 3 N HCl, 3 N KOH. Nutrient Agar: Beef Extract: Peptone: Agar: 0.3% 0.5% 1.5%

PROCEDURE: 1. You will be making 200 ml of Nutrient Agar. To weigh out Beef Extract, first tare a tonguedepressor, then dip it into the Beef Extract and weigh. Adjust the amount of Beef Extract until the correct amount is obtained. Be sure to be careful not to get Beef Extract on to the balance! You need to weight out enough Beef Extract to get a 0.3% solution. Place the tongue depressor into the flask, beef extract side down. 2. Tare a weigh boat and weigh out enough Peptone and add that to the flask 3. Add 200 ml of distilled water and swirl to dissolve the peptone and beef extract. Check thepH, it should be 7.0. 4. Tare a weigh boat and weigh out enough Agar and add that to the flask.

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5. With a bunsen burner, tripod, asbestos wire-gauze, heat the medium to boiling to dissolve theagar. CAREFUL: 1) keep the rotating the flasks to prevent the agar from cooking onto thebottom of the flask and 2) watch out: boiling agar can froth and boil out all over the lab bench.As soon as it begins to boil take it off the heat and put it on to the bench. Allow it to cool a fewminutes. 6. While the agar is still warm, but not hot, pipette 3 ml each into 4 13x100 mm screw cap culture tubes. 7. Label the flask and your tubes with your name. 8. After preparation of your medium, the instructor will take you to the autoclave. 9. Place your media in the autoclave with those of the rest of the class. 10. After discussion of the parts of the autoclave, autoclave the medium for 20 minutes. 9.2 Preparation of antibiotic stock solutions Antibitiotics may be received as powders or tablets. It is recommended to obtain pure antibiotics from commercial sources, and not use injectable solutions. Powders must be accurately weighed and dissolved in the appropriate diluents (Annexure III) to yield the required concentration, using sterile glassware. Standard strains of stock cultures should be used to evaluate the antibiotic stock solution. If satisfactory, the stock can be aliquoted in 5 ml volumes and frozen at -20ºC or -60ºC.

Stock solutions are prepared using the formula (1000/P) X V X C=W, where P+potency of the anitbiotic base, V=volume in ml required, C=final concentration of solution and W=weight of the antimicrobial to be dissolved in V.

Preparation Of Antibiotic Stock Solutions
Ampicillin: Stocks & Usagez

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? ? ?

Stock Concentration - 50mg/ml in H2O Aliquots - 100?l and 500?l Working Concentration - 50?g/ml

Preparation of 80ml stock solution
? ? ? ? ? ? ? ? ?

Ampicillin is kept in the 4C fridge in 68-564D. It is light sensitive. Weigh 4g of ampicillin into a small weigh boat. Add 80ml of milliQ to a 250ml bottle. Add the ampicillin to the milliQ Mix/vortex so all the ampicillin goes into solution. Filter sterilize the solution into a falcon tube using a 20ml syringe and a 200nm filter. Aliquot into pcr tubes and 1.7ml eppendorfs. Store at -20C. Store the small aliquots in the small box and the big aliquots in the larger box.

Tetracycline:
Stocks & Usage
? ? ?

Stock Concentration - 5mg/ml in 70% Ethanol Aliquots - 200?l and 1ml Working Concentration - 20?g/ml

Preparation of 80ml stock solution
? ? ? ? ? ? ? ? ? ?

Tetracycline is kept in the 4C fridge in 68-564D. It is light sensitive. Weigh 400mg of tetracyline HCL into a small weigh boat. Dilute 95% Ethanol to 70% using milliQ water. o Adding 20ml of milliQ to 60ml of 95% ethanol gives 80ml of 71% ethanol. Add 80ml of 70% Ethanol to a 250ml bottle. Add the tetracycline HCL to the ethanol. Mix/vortex vigorously so all the tetracycline goes into solution. Filter sterilize the solution into a falcon tube using a 20ml syringe and a 200nm filter. Aliquot into pcr tubes and 1.7ml eppendorfs. Store at -20C and protect any unused stock solution from light. Store the small aliquots in the small box and the big aliquots in the larger box.

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Streptomycin:
Stocks & Usage
? ? ?

Stock Concentration: 50mg/ml in H2O Aliquots - 0.5 ml Working Concentration - 10-50?g/ml

Preparation of 40ml stock solution
? ? ? ? ? ? ? ?

Streptomycin is kept in the 4C fridge in 68-564D. I don't think it is light sensitive (not stored in opaque bottle). Weigh 2g of streptomycin into a small weigh boat. Add 40ml of milliQ to a 100 or 250ml bottle. Add the streptomycin to the milliQ Mix/vortex so all the streptomycin goes into solution. Filter sterilizes the solution into a falcon tube using a 60ml syringe and a 200nm filter. Aliquot into 1.7ml eppendorfs. Store at -20C.

9.3 Preparation of dried filter paper Antibiotics discs: Whatman filter paper no. 1 is used to prepare discs approximately 6 mm in diameter, which are placed in a Petri dish and sterilized in a hot air oven.

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The loop used for delivering the antibiotics is made of 20 gauge wire and has a diameter of 2 mm. This delivers 0.005 ml of antibiotics to each disc. Prepare for 0.1 and 0.2 and 0.3 and 0.4 and 0.5 concentration discs prepare for Antibiotic stock solution using different types of micro pipits 9.4 Storage of commercial antimicrobial discs Cartridges containing commercially prepared paper disks specifically for susceptibility testing are generally packaged to ensure appropriate anhydrous conditions. Discs should be stored as follows: * Refrigerate the containers at 8?C or below, or freeze at -14?C or below, in a nonfrost-free freezer until needed. Sealed packages of disks that contain drugs from the ß-lactam class should be stored frozen, except for a small working supply, which may be refrigerated for at most one week. Some labile agents (e.g., ampicilline, tetracycline, and penicillin,

streptomycin combinations) may retain greater stability if stored frozen until the day of use. * The unopened disc containers should be removed from the refrigerator or freezer one to two hours before use, so they may equilibrate to room temperature before opening. This procedure minimizes the amount of condensation that occurs when warm air contacts cold disks. * Once a cartridge of discs has been removed from its sealed package, it should be placed in a tightly sealed, desiccated container. When using a disc-dispensing apparatus, it should be fitted with a tight cover and supplied with an adequate desiccant. The dispenser should be allowed to warm to room temperature before opening. Excessive moisture should be avoided by replacing the desiccant when the indicator changes color. * When not in use, the dispensing apparatus containing the discs should always be refrigerated. * Only those discs that have not reached the manufacturer's expiration date stated on the label may be used. Discs should be discarded on the expiration date.

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A) Glass plate and wet Antibiotic B) Dried Antibiotic disc for Concentration for Disc 9.5 Preparation of E.coli Culture:
Luria-Bertani (LB) media (1 L): Mix 10 g of Bacto-tryptone, 5 of Yeast extract, and 10 g of NaCl (for taste). pH to 7.5 w/ NaOH. And dH2O to 1 L (Autoclave) Mix 500 mL of LB media with 7 g of Agar (Autoclave). Cool to ~55-65oC prior to pouring. The addition of antibiotics should be made before pouring and at a temperature not higher than 55oC. Antibiotics can also be spread on previously made plates, but this is not very effective (unequal absorption, etc...)

0.1and 0.2 discs

Procedure:
1.Streak E.coli cells (DH5?, HB101, GM8) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol) 2.Allow cells to grow at 37oC overnight 3.Place one colony in 10 mL LB media (+antibiotic selection if necessary), grow overnight at 37oC

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4.Take 2 ml LB media and save for blank. Transfer 5 mL overnight DH5? culture into 500 mL LB media in 3 L flask 5. Allow cell to grow at 37oC (250 rpm), until OD600= 0.4 (~2-3 hours) 6. Transfer cells to 2 centrifuge bottles (250 mL), and place cells on ice for 20 mins 7. Centrifuge cells in Sorval GSA rotor at 4oC for 10 mins at 3,000 gSubsequent resuspensions may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room 8. Pour off media and resuspend cells in 30 mL of cold 0.1 M CaCl2. Transfer the suspended cells into 50 mL polypropylene falcon tubes, and incubate on ice for 30 mins 9. Centrifuge cells using Sorval RT6000B rotor at 4oC for 10 mins at 3,000 g (2500 rpm) 10.Pour supernatant and resuspend cells (by pipetting) in 8 mL cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 ?L into (1.5 mL) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months 11.through the process, cells should be treated with care. No vortexing or excess pipetting should be performed, specially when the cells have been resuspended in CaCl2 because lysis will result, decreasing the amount of competent cells). Also, depending on the density of the cells, higher or lower volumes CaCl2 can be used to increase the concentration of cells per tube.
9.6 Preparation of inoculum 1. Using a sterile inoculating loop or needle, touch four or five isolated colonies of the organism to be tested. 2. 3. Suspend the organism in 2 ml of sterile saline. Vortex the saline tube to create a smooth suspension.

4. Adjust the turbidity of this suspension to a 0.5 E.coli standard by adding more organism if the suspension is too light or diluting with sterile saline if the suspension is too heavy. 5. Use this suspension within 15 minutes of preparation.

Inoculum preparation Organisms to be tested must be in the log phase of growth in order for results to be valid. It is recommended that subcultures of the organisms to be tested be made the previous day. Never use extremes in inoculum density. Never use undiluted overnight broth cultures or other unstandardized inocula for inoculating plates.

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Effect of antibiotics on growth curve by using micro organisms

If the organism is difficult to suspend directly into a smooth suspension, the growth method of preparing the inoculums should be used. However, the recommended organisms listed in this procedure all produce smooth suspensions with little difficulty. See the Clinical Laboratory Standards Institute document (3) for the growth procedure method for preparing the inoculums, if needed. 9.7 Inoculation of the nutrient Agar plates: 1. Dip a sterile swab into the inoculum tube.

2. Rotate the swab against the side of the tube (above the fluid level) using firm pressure, to remove excess fluid. The swab should not be dripping wet 3. Inoculate the dried surface of a nutrient agar plate by streaking the swab three times over the entire agar surface; rotate the plate approximately 60 degrees each time to ensure an even distribution of the inoculum 4. 5. Rim the plate with the swab to pick up any excess liquid Discard the swab into an appropriate container.

6. Leaving the lid slightly ajar, allow the plate to sit at room temperature at least 3 to 5 minutes, but no more than 15 minutes, for the surface of the agar plate to dry before proceeding to the next step.

Placement of the antibiotic disks 1. Place the appropriate antimicrobial-impregnated disks on the surface of the nutrient agar, using either forceps to dispense each antimicrobial disk one at a time, or a multidisk dispenser to dispense multiple disks at one time. (See steps a. through d. for the use of the multi-disk dispenser or steps e. through g. for individual disk placement with forceps. a. To use a multidisc dispenser, place the inoculated nutrient agar plate on a flat surface and remove the lid

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Effect of antibiotics on growth curve by using micro organisms

b. Place the dispenser over the agar plate and firmly press the plunger once to dispense the disks onto the surface of the plate. c. Lift the dispenser off the plate and using forceps sterilized by either cleaning them with an alcohol pad or flaming them with isopropyl alcohol, touch each disk on the plate to ensure complete contact with the agar surface. This should be done before replacing the Petri dish lid as static electricity may cause the disks to relocate themselves on the agar surface or adhere to the lid d. Do not move a disk once it has contacted the agar surface even if the disk is not in the proper location, because some of the drug begins to diffuse immediately upon contact with the agar. e. To add disks one at a time to the agar plate using forceps, place the Agar plate on the template provided in this procedure Sterilize the forceps by cleaning them with a sterile alcohol pad and allowing them to air dry or immersing the forceps in alcohol then igniting. f. Using the forceps carefully remove one disk from the cartridge

g. Partially remove the lid of the Petri dish. Place the disk on the plate over one of the dark spots on the template and gently press the disk with the forceps to ensure complete contact with the agar surface. Replace the lid to minimize exposure of the agar surface to room air h. Continue to place one disk at a time onto the agar surface until all disks have been placed as directed in steps f. and g. above. 2. Once all disks are in place, replace the lid, invert the plates, and place them in a 35°C air incubator for 16 to 18 hours. When testing Staphylococcus against oxacillin or vancomycin, or Enterococcus against vancomycin, incubate for a full 24 hours before reading

1. Paper antibiotic discs 0.1, and 0.3 agar

2. Placement of antibiotic discs from nutrient

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Effect of antibiotics on growth curve by using micro organisms

10. Antibiotic Sensitivity Tests:
Which antibiotics are most effective against gram negative bacteria (E. coli) Materials and Supplies see order sheet) Inoculating loop Bunsen burner or propane torch Microscope slides Petri dishes Microscope (10, 40, 100 objectives +10X eyepiece,200 objectives ) Grease pencils E. coli culture Antibiotic disks (treated with tetracycline, streptomycin, penicillin, ampicillin, or other) Grade Level Expectations: ? ? ? ? 1.1.6. Analyze structural, cellular, biochemical, and genetic characteristics in order to determine the relationships among organisms 1.2.6 Understand cellular structures, their functions, and how specific genes regulate these functions 2.1.2 Understand how to plan and conduct systematic and complex scientific investigations 2.1.3 Synthesize a revised scientific explanation using evidence, data, and inferential logic.

Protocol: Part 1. You will place a small paper disk treated with each test compound in one quadrant of a plate of nutrient agar that contains the bacterium. If the compound is effective, a clear ring of no bacterial growth (zone of inhibition) appears around the disk after the bacterial plate is incubated.

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Effect of antibiotics on growth curve by using micro organisms

Procedure: Use aseptic technique at all times while handling these plates 1. Your group will receive 4 petri dishes of nutrient agar 2. Write your names and lab period on the lid of the plate. Also label the Petri dishes #1 and #2and#3and#4 3. Turn over the dishes and use a grease pencil to draw a line down the middle, turn the plate 90 degrees and draw another line so that the line is an + shape. This divides the plate into quarters or quadrants. 4. Inoculate the agar of plate #1 with bacteria specimen #1. Be sure you are using aseptic technique (hold the plate and clamshell the plate, do not take the cover off, simply lift the edge of the plate and insert the inoculating loop). Streak the plate in a zig zag motion being sure to cover the entire plate with the bacterium 5. Repeat #4 with bacteria specimen #2. 6. Obtain the four antibiotic disks (make sure they are all different). Use forceps to place them in the center of each quadrant. The letters on the disks identify which antibiotic they contain. 7. Be sure disks adhere to the agar, do not push them into the agar. 8. Place in a 37 degree Celsius oven for 2-3 days.

Part 2 (2-3 days after placing plates in oven) 1. Observe the zones of inhibition on both plates. 2. Measure the diameter of the zones of inhibition and record in mm. Record data in table. 3. Use a sterile inoculating loop (pass through flame) and take a sample of bacteria by placing a small amount on the loop. Take a sample and streak a small area of a slide. Do this for e.coli bacteria samples.

4. Observe under a microscope beginning with the lowest power and working up to 100X objective (1000X magnification with a 10X objective) using oil on the 100X magnification

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Effect of antibiotics on growth curve by using micro organisms

11. Incubation of the plates:
A temperature range of 35°C ± 2°C is required. Do not incubate plates in CO2 as this will decrease the pH of the agar and result in errors due to incorrect pH of the media. Results can be read after 18 hours of incubation unless you are testing Staphylococcus against oxacillin or vancomycin, or Enterococcus against vancomycin. Read the results for the other antimicrobial disks then reincubate the plate for a total of 24 hours before reporting vancomycin or oxacillin.

12. Measuring zone sizes:
1. Following incubation, measure the zone sizes to the nearest millimeter using a ruler or caliper; include the diameter of the disk in the measurement 2. When measuring zone diameters, always round up to the next millimeter.

3. All measurements are made with the unaided eye while viewing the back of the petri dish. Hold the plate a few inches above a black, nonreflecting surface illuminated with reflected light 4. View the plate using a direct, vertical line of sight to avoid any parallax that may result in misreading. 5. Record the zone size on the recording sheet.

6. If the placement of the antibiotic disk or the sizes 0.1 to 0.5 mm of the zone does not allow you to read the diameter of the zone, measure from the center of the disk to a point on the circumference of the zone where a distinct edge is present (the radius) and multiply the measurement by 2 to determine the diameter 7. Growth up to the edge of the disk can be reported as a zone of 0 mm.

8. When measuring the zone of inhibition for organisms that swarm (e.g.,e.coli sp.), ignore the thin veil of swarming growth in an otherwise obvious zone of inhibition.

9. Organisms such as Proteus mirabilis, which swarm, must be measured differently than nonswarming organisms. Ignore the thin veil of swarming and measure the outer margin in an otherwise obvious zone of inhibition.

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10. Distinct, discrete colonies within an obvious zone of inhibition should not be considered swarming. These colonies are either mutant organisms that are more resistant to the drug being tested, or the culture was not pure and they are a different organism. If it is determined by repeat testing that the phenomenon repeats itself, the organism must be considered resistant to that drug.

13. Result:
Effect of antibiotics studies of E. coli cultures exposed to four suprainhibitory Concentrations of ampicillin and penicillin and tetracycline and streptomycin growth curves E. coli expose from Ampicillin growth curve first increase after curve is stationary after time 180 minutes from reduced from growth curve Penicillin growth curves first increase after curve is stationary phase Time 120 minutes continues from growth curve Tetracycline growth curves first increase after curve is stationary after time 150 minutes from reduced from growth curve Streptomycin growth inhibition stating from curve is stationary phase after time 120 minutes from reduced from growth curve Effect of antibiotics resistance and antibiotic sensitivity studies from four antibiotics of zone of inhibition formed in Petri discs

Data Tables: Antibiotics Zone diameter for Plate 1 (mm) 6 mm --14 mm 3 mm Zone diameter Plate #2 (mm) 7 mm 6 mm 15.5 mm --Zone diameter Zone diameter for Plate #3 for Plate #4 (mm) (mm) 8.5 mm 8 mm 18 mm ---5.5 mm 10 mm 20 mm --Zone diameter for Plate #5 (mm ---11.5 mm -------

Ampicillin Penicillin Tetracycline Streptomycin

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Ampcillin Antibiotic Disics :

(0.1 ml)

(0.2 ml)

( 0.3 ml)

( 0.4 ml)

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Penclin Antibiotic Discs

(0.2 ml)

( 0.3ml)

( 0.4ml)

(0.5ml)

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Tetracycline Antibiotic Discs:

( 0.1ml )

( 0.2ml )

(0.3ml)

(0.4ml)

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Effect of antibiotics on growth curve by using micro organisms

Sterptomycine Antibiotic Discs

( 0.1ml)

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S.NO 1 2 3 4 5 6 7 8 TIME OD values 30 0.05 60 0.1 90 0.124 120 0.68 150 0.68 180 0.64 210 0.136 240 0.116

TABLES:

FIG NO 1

Growth curve for 0.1ml Amphillicin
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 50 100 150 Time, minutes 200 250 300

OD at 600 nm

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S.NO 1 2 3 4 5 6 7 8 FIG NO 2

Time 30 60 90 120 150 180 210 240

OD values 0.056 0.136 0.173 0.165 0.16 0.155 0.146 0.136

Growth curve for 0.2ml Amphillicin
0.2 0.18 0.16 0.14 OD at 600nm 0.12 0.1 0.08 0.06 0.04 0.02 0 0 1 2 3 4 5 Time, minutes 6 7 8 9 10

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD values 0.04 0.158 0.178 0.19 0.196 0.196 0.154

FIG NO 3

0.3 ml Amphicillin at 600 nm growth curve
0.25

0.2

OD at 600 nm

0.15

0.1

0.05

0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD values 0.06 0.229 0.26 0.269 0.27 0.282 0.261

FIG NO 4

0.4 ml Amphicillin at 600 nm growth curve

0.3 0.25 0.2 0.15 0.1 0.05 0 0 50 100 150 Time minutes 200 250

OD at 600 nm

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.09 0.259 0.28 0.29 0.295 0.304 0.283

FIG NO 5

0.5 ml Amphicillin at 600 nm growth curve
0.35 0.3 OD at 600 nm 0.25 0.2 0.15 0.1 0.05 0

0

50

100 150 Time minutes

200

250

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S.NO 1 2 3 4 5 6 7 8 FIG NO 6

Time 30 60 90 120 150 180 210 240

OD 0.001 0.3 0.35 0.36 0.37 0.37 0.169 0.14

0.1 Ml Pencillin at 600 nm growth curve
0.45 0.4 0.35

OD at 600 nm

0.3 0.25 0.2 0.15 0.1 0.05 0 0 50 100 150 200 250 300

Time minutes

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S.NO 1 2 3 4 5 6 7 8

Time 30 60 90 120 150 180 210 240

OD 0.001 0.3 0.31 0.312 0.314 0.316 0.169 0.12

FIG NO 7

0.2 Ml Pencillin at 600 nm growth curve
0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 50 100 150 200 250 300

OD at 600 nm

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.05 0.301 0.31 0.311 0.312 0.32 0.315

FIG NO 8

0.3 Ml Pencillin at 600 nm growth curve
0.35
0.3 0.25 OD at 600 nm 0.2

0.15
0.1 0.05 0 0 50

Time minutes 100 150

200

250

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.07 0.249 0.292 0.277 0.289 0.3 0.411

FIG NO 9 0.4 Ml Pencillin at 600 nm growth curve
0.45 0.4 0.35

OD at 600 nm

0.3

0.25 0.2

0.15 0.1 0.05 0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.1 0.33 0.343 0.345 0.35 0.385 0.359

FIG NO 10

0.5 Ml Pencillin at 600 nm growth curve
0.45 0.4 0.35 0.3

OD at 600 nm

0.25 0.2 0.15 0.1 0.05 0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7 8

Time 30 60 90 120 150 180 210 240

OD 0.001 0.12 0.154 0.16 0.162 0.164 0.168 0.15

FIG NO 11

0.1 ml Tetracycline growth curve
0.18 0.16 0.14 0.12

OD at 600 nm

0.1 0.08 0.06 0.04 0.02 0 0 50 100 150 200 250 300

Time minutes

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S.NO Time
1 2 3 4 5 6 7 8 30 60 90 120 150 180 210 240

OD 0.001 0.2 0.23 0.24 0.25 0.251 0.169 0.14

FIG NO 12

0.2 ml Tetracycline growth curve
0.3

0.25

0.2

OD at 600 nm

0.15

0.1

0.05

0 0 50 100 150 200 250 300

Time minutes

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S.NO TIME 1 30 2 60 3 90 4 120 5 150 6 180 7 210

OD 0.15 0.284 0.295 0.294 0.169 0.150 0.143

FIG NO 13

0.3 Ml Tetracycline growth curve
1.2

1

OD at 600 nm

0.8

0.6

0.4

0.2

0 0 50 100 150 200 250

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Effect of antibiotics on growth curve by using micro organisms

S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.161 0.278 0.29 0.293 0.16 0.151 0.145

FIG NO 14

0.4 Ml Tetracycline at 600 nmgrowth curve
0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 50 100 150 200 250 TIME minutes

OD at 600 nm

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.11 0.276 0.282 0.285 0.166 0.152 0.150

FIG NO 15

0.5 Ml Tetracycline at 600nm growth curve
0.35 0.3 0.25

OD at 600 nm

0.2

0.15 0.1

0.05 0 0 50 100 150 200 250

Time minutes

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S.NO
1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.0231 0.257 0.248 0.25 0.215 0.227 0.233

FIG NO 16

0.1 Ml Streptomycin at 600 nm growth curve

0.3 0.25 0.2

OD at 600 nm

0.15 0.1 0.05 0 0 50 100 150 200 250

Time minutes

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S.NO
1 2 3 4 5 6 7

Time 30 60 90 120 150 180 210

OD 0.41 0.456 0.458 0.458 0.453 0.454 0.452

FIG NO 17

0.2 Ml Streptomycin at 600 nm growth curve
0.47

0.46

0.45

OD at 600nm

0.44

0.43

0.42

0.41

0.4 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.661 0.988 0.989 0.931 0.603 0.600 0.575

FIG NO 18

0.3 Ml Streptomycin at 600 nm growth curve
1.2 1 0.8 0.6 0.4 0.2 0 0 50 100 Time minutes 150 200 250

OD at 600 nm

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.621 0.813 0.838 0.848 0.544 0.500 0.470

FIG NO 19

0.4 Ml Streptomycin at 600 nm growth curve

1 0.9 0.8

OD at 600 nm

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

OD 0.693 1.054 1.075 1.055 0.669 0.550 0.450

FIG NO 20

0.5 Ml Streptomycan NTU growth curve
1.2

1

NTU values

0.8

0.6

0.4

0.2

0 0 50 100 150 200 250

time minutes

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Effect of antibiotics on growth curve by using micro organisms

S.NO 1 2 3 4 5 6 7

Time 30 60 90 120 150 180 210

NTU 15 32 35 36 37 38 30

FIG NO 21

0.1 Ml Tetracycline NTU growth curve
45 40 35 30

NTU values

25 20 15 10 5 0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

Time 30 60 90 120 150 180 210

NTU 20 55 58 60 50 40 30

FIG NO 22

0.2ml Tetracycline NTU growth curve
70 60

NTU values

50 40 30 20 10 0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7 FIG NO 23

TIME 30 60 90 120 150 180 210

NTU 61.9 98.3 97.7 90.1 62.8 55.5 30.3

0.3 Ml tetracycline NTU growth curve
120

100

NTU values

80

60

40

20

0 0 50 100 150 200 250

time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 55.3 81.3 93.6 98.3 85.5 80.3 75.3

FIG NO 24

0.4 Ml tetracycline NTU growth curve
1.2

1

NTU values

0.8

0.6

0.4

0.2

0 0 50 100 150 200 250

time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 152 275 308 315 310 285 250

FIG NO 25

0.5 Ml Tetracycline NTU growth curve
350 300 250 200 150 100 50 0 0 50 100 150 200 250

NTU values

Time minutes

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S.NO 1 2 3 4 5 6 7 8

Time 30 60 90 120 150 180 210 240

NTU 20 34 45 60 68 68 68 60

FIG NO 26

0.1ml Streptomycin NTU Growth Curve
80 70

NTU values

60 50 40 30 20 10 0 0 50 100 150 200 250

Time minutes
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S.NO 1 2 3 4 5 6 7 8

Time 30 60 90 120 150 180 210 240

NTU 20 34 45 60 80 80 80 60

FIG NO 27

0.2ml streptomycin NTU Growth curve
90 80 70

NTU values

60 50 40 30 20 10 0 0 50 100 150 200 250 300

Time minutes
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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 182.1 193 198 199.7 200 199.7 184

FIG NO 28

205

0.3 Ml Streptomycin NTU growth curve

200

NTU values

195

190

185

180 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 186.5 188 198.5 199 200 199.5 190

FIG NO 29

0.4 Ml Streptomycin NTU growth curve
202 200

NTU values

198 196 194 192 190 188 186 184 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 516 537 540 550 564 554 530

FIG NO 30

0.5 Ml tetracycline NTU growth curve
1.2

1

0.8

NTU values

0.6

0.4

0.2

0 0 50 100 150 200 250

time minutes
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S.NO 1 2 3 4 5 6 7

Time 30 60 90 120 150 180 210

NTU 33 38 67 70 75 76 50

FIG NO 31

0.1ml Penicillin NTU Growth Curve
90 80 70 60 NTU Values 50 40 30 20 10 0 0 50 100 Timeminutes 150 200 250

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S.NO 1 2 3 4 5 6 7

Time 30 60 90 120 150 180 210

NTU 23 45 56 66 70 75 70

FIG NO 32

80 70 60 50 NTU Values 40 30 20 10 0 0 50

0.2ml Pencillin NTU Growth Curve

100

150

200

250

Timeminutes

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S.NO 1 2 3 4 5 6 7 FIG NO 33 TIME 30 60 90 120 150 180 210 NTU 64.3 60.6 100.5 102.5 172 240 251

0.3 Ml pencilline NTU growth curve
300

250

NTU values

200

150

100

50

0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 93.4 94.5 92.5 89.5 215 249 189

FIG NO 34

0.4 Ml pencilline NTU growth curve
300 250

NTU values

200 150 100 50 0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 265 273 285 286 285 290 286

FIG NO 35

0.5 Ml pencilline NTU growth curve
295

290

NTU values

285

280

275

270

265

260 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7 8

Time 30 60 90 120 150 180 210 240

NTU 20 38 45 55 67 67 68 60

FIG NO 36

0.1ml Amphicillin NTU Growth Curve
80 70 60 50

NTU values

40 30 20 10 0 0 50 100 150 200 250 300

Time minutes
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S.NO
1 2 3 4 5 6 7 8

Time 30 60 90 120 150 180 210 240

NTU 42 55 60 110 110 112 115 100

FIG NO 37

140

0.2ml Amphillicin NTU Growth Curve

120

100

NTU VALUES

80

60

40

20

0 0 50 100 150 200 250 300

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 14.7 37.2 38 39.4 102 105 108

FIG NO 38

0.3 Ml Amphicillin NTU growth curve
120

100

NTU values

80

60

40

20

0 0 50 100 150 200 250

Time minutes

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S.NO 1 2 3 4 5 6 7

TIME 30 60 90 120 150 180 210

NTU 20 44.3 46 48.9 74 126 132

FIG NO 39

0.4 Ml Amphicillin NTU growth curve
140 120

NTU values

100 80 60 40 20 0 0 50 100 150 200 250

Time minutes
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S.NO TIME 1 30 2 60 3 90 4 120 5 150 6 180 7 210

NTU 32.1 84 160 169 170 178 150

FIG NO 40

0.5 Ml Amphicillin NTU growth curve
200 180 160 140 120 100 80 60 40 20 0 0 50 100 150 200 250

NTU values

Time minutes

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comparision of different antibiotics on growth of micro-organisms
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Amphicillin Pencillin Tetracycline Streptomycin

concentration

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Concentration v/s different antibiotics
25

Concentration(ml)

20 15 10 5 0 8.5 7 6 5.5 6 11.5 10 8

20 18 15.5 14 0.1mL

o.2mL
3 0.3mL 0.4mL 0.5mL

Different antibiotics

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Effect of antibiotics on growth curve by using micro organisms

Conclusion:
Antibiotics are prescribed for illnesses caused by bacteria, not by viruses. Prescriptions are written to cover the time needed to help your body fight all the harmful bacteria. If you stop your antibiotic early, the bacteria that have not yet been killed can restart an infection. If you take antibiotics unnecessarily you may contribute to the development of antibiotic resistance. As seen in this study bacterial infections may or may not be cured with one dose of antibiotics. Hospital laboratories use similar techniques to determine bacterial susceptibility to different antibiotics. Effect of antibiotics on tetracycline is high concentration compare to other three antibiotics (ampcillin.pencilline, streptomycin) Effect of antibiotics on growth curve penicillin is high growth inhibition

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4 . Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 36:493-496 .5. Clinical Laboratory Standards Institute. 2006. Performance standards for antimicrobial disk susceptibility tests; Approved standard—9th ed. CLSI document M2-A9. 26:1. Clinical Laboratory Standards Institute, Wayne, PA .6. Difco. 1984. Difco manual, 10th ed. Difco Laboratories, Detroit, MI

.7. Kirby, W. M. M., G. M. Yoshihara, K. S. Sundsted, and J. H. Warren. 1957. Clinical usefulness of a single disc method for antibiotic sensitivity testing. Antibiotics Annu. 19561957:892. 8. Winn, Jr., W., et al. 2006. Konemann’s color atlas and diagnostic text of microbiology, 6th ed., p. 945–1021. Lippencott Williams & Wilkins Publishers, Philadelphia, PA. 9. Jorgensen, J. H., and J. D. Turnidge. 2007. Susceptibility test methods: dilution and disk diffusion methods, p. 1152–1172. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry, and M. A. Pfaller (ed.), Manual of clinical microbiology 9th ed. ASM Press, Washington, D.C.
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Websites:
13. www.wekipedia.com

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