Sweet hopes for diabetics Good news for diabetics.

Sweet hopes for diabetics Good news for diabetics.

A three-year study carried out by Universiti Teknologi Malaysia in Skudai has confirmed previous findings that cinnamon has the potential to lower sugar levels.

UTM research and development manager Prof Dr Mohammad Roji Sarmidi said their research showed that the spice, known as kayu manis locally, has positive effects on the disease, especially Type II diabetes.
Type II diabetes causes cells to lose their ability to respond to insulin, the hormone that tells the body to remove excess glucose from the bloodstream. This condition usually develops in people in their middle age and prematurely kills an estimated 100 million of the world’s population every year. Dr Mohammad Roji said herbalists all over the world had used cinnamon in the treatment of diarrhoea and arthritis, as cinnamon extract was found to improve blood circulation, heal wounds, reduce pain spasm and prevent ulcer and allergies.
“In the last decade, laboratory studies have also revealed that cinnamon extract mimicked insulin action in the cells,” he said. Insulin regulates glucose metabolism, helping body cells to convert glucose to energy and keep blood sugar levels normal. “Studies by the Agriculture Research Service in the United States have also found that certain substances in cinnamon helps cells become more responsive to insulin,” he added. Cinnamon is an ingredient used in cooking, and in cakes, pastries and beverages like coffee and tea.
Dr Mohammad Roji said UTM would conduct further studies next year, which would cover tests on animals and metabolic profiling for diabetic patients.

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Hybridoma - a technique for eternal production of monoclonal antibodies in cell cultures

An explanation of the diversity of the immune system
The most important task for the immune system is to defend the body against bacteria, virus and other microorganisms. The specific defense is exerted by a subgroup of white blood cells (lymphocytes). The immune system needs to recognize and react specifically with a large number of foreign substances (antigens). How the lymphocytes develop these vital properties and how they build up the highly specialized recognition system of the immune apparatus has long been an area of intensive research. pharmaguideline.
Niels K. Jerne is the leading theoretician in immunology during the last 30 years. In three main theories he has elucidated central issues concerning specificity, development and regulation of the immune system in a comprehensive and convincing way. By his theories Jerne has outlined the development of modern immunology.
Theory 1: Specificity is predeterminedIn his Natural-Selection
Theory of Antibody Formation from 1955 Jerne explains the development of a specific antibody response in the following way. Each individual has a large number of natural antibodies with specificities for all antigens towards which the individual can respond. These antibodies develop already during fetal life in the absence of external antigens. The foreign antigen then selects the antibody molecule which has the best fit. The antigen-antibody binding stimulates the production of this particular antibody specificity.Jerne's natural-selection theory contrasted to the dogmatic views of the antibody response as formulated in the instruction theories which were prevailing at that time. According to these theories the antigen serves a template for the production of antibodies.In Jerne's natural selection theory it is implied that the generation of the enormous number of antibody specificities is independent of exogenous antigens. This view on the nature of the immune system constitutes the basis for modern immunology.
--->Regulatory affairs in pharmaceutical <---- Theory 2: Reactivity against self-antigens creates diversity
The natural-selection theory is mainly concerned with the maturation of the immune system after it has acquired the ability to react with antigen. In the second theory on the Somatic Generation of Immune Recognition set forth in 1971 Jerne explains how the immune system develops from stem cells to mature lymphocytes which can react with antigen. He presupposes that every individual possesses all genes needed for the production of antibodies, and antibody-like molecules, which can bind all strong transplantation antigens of the species. Jerne suggests that lymphocytes mature in the thymus gland and in other lymphoid organs where they are exposed to the transplantation antigens of the individual. Cells which recognize the antigens are stimulated and enter cell division. As mutations accumulate in rapidly dividing cells new immunological specificities may develop. At the same time the specificities of the lymphocytes for self transplantation antigens are weakened. The mature lymphocytes will recognize foreign antigen associated with transplantation antigens. The theory explains how the immune system normally matures through the influence of self antigens. It also offers an explanation for the regulation of immunological specificity by genes belonging to the transplantation system.
Theory 3. Antibodies, anti-anti-bodies ...In his third main theory, the Network Theory from 1974, Jerne explains how the specific immune response is regulated. The theory has greatly stimulated research and led to new insights into the immune system. Recently its principles have been applied to diagnosis and treatment of disease.
A basis for the network theory was the observation that antibodies can elicit anti-antibodies directed against antigen binding structures on the first antibody .Moreover, anti-antibodies can stimulate the production of still another generation of antibodies, anti-anti-antibodies. Essentially, this antibody cascade is endless successively adding new specific properties to the immune system. The various antibody generations either stimulate or suppress the production of one another. Under normal conditions the network is balanced. When an antigen is introduced the equilibrium is disturbed. The immune system tries to restore balance which leads to an immune response against the antigen.1. Infectious diseases. Anti-antibodies have been used in animals as a kind of vaccine against parasitic infections (trypanosomiasis), urinary tract infections, hepatitis and other infectious diseases.
2. Allergy. Anti-pollen antibodies may elicit allergic symptoms when an allergic person is exposed to pollen. The production of anti-pollen antibodies has been prevented in animals by anti-antibodies.
3. Autoimmune disease. Autoimmune disease may be caused by antibodies directed against the body's own tissues. Experimental autoimmune disease has been successfully treated with anti-antibodies.
4. Transplantation. Anti-antiimmunity may be important in organ transplantation by contributing to immunological tolerance against antigen on the foreign graft.
5. Endocrinology. Anti-antibodies against hormones and hormone receptors may prevent binding of the hormone to the receptors. This has been described for insulin and its receptor.
6. Tumours. Anti-antibodies have been attempted as treatment of certain tumours of the human immune system.
--->Pharmaceutical Regulatory affairs <---- Hybridoma - a technique for eternal production of monoclonal antibodies in cell cultures
Besides gene technology, which has already been honoured by several Nobel Prizes, the hybridoma technique represents the most important methodological advance within the field of biomedicine during the 1970s. The development of this technique is based on several observations concerning basic biological phenomena.
There are cells in the body - immune lymphocytes - which can produce millions of different antibodies. However, each single cell can only produce antibodies with a certain predetermined specificity. A prerequisite for the formation of a multitude of antibodies is, therefore, the existence of an excess of lymphocytes. If the body is exposed to a certain foreign antigen there may be stimulation of a lymphocyte which fortuitously has been endowed with the capacity to identify this particular antigen. This lymphocyte then starts to divide and forms a clone of cells which produces identical - monoclonal - antibodies.
The development of a clone of cells in connection with a normal immune response occurs under carefully controlled conditions. In rare cases, however, the body loses control over a clone of antibody producing cells. This may lead to formation of a special type of tumour (myeloma). Myeloma cells usually retain their capacity to produce a certain antibody, but because of the accidental emergence of the tumour one normally does not know with which antigen this antibody reacts.
White blood cells responsible for producing antibodies are highly specialized cells. As a consequence they lack capacity to survive for a longer time if they are removed from the body and incubated in a tissue culture medium. In contrast, myeloma cells can occasionally be cultivated continuously. Since long, biomedical research workers have nourished the dream to be able to propagate clones of cells which produce antibodies with predetermined specificity. This dream materialized when Georges J.F. Köhler and César Milstein in 1975 introduced the so-called hybridoma technology for production of monclonal antibodies. The principle features of the hybridoma technology is as follows pharma .blogspot.com
Figure 2. Principle steps in the production of a hybridoma. Spleen cells are prepared from animals, usually mice, which have been immunized with a selected antigen. These cells are then fused with myeloma cells maintained in culture in the laboratory. The product of this fusion is referred to as a hybridoma. Surprisingly, a hybrid of two cells can survive and also continue to divide. In this particular hybrid the myeloma cells contribute the capacity for survival, whereas the spleen cells direct the synthesis of antibodies with the preselected specificity. By special arrangements it is possible to achieve a multiplication of hybridoma cells but not of isolated myeloma cells. The hybrids obtained are propagated in a highly diluted state so that colonies deriving from single hybrid cells can be isolated. By use of a sensitive method the clones which produce the specific antibodies are identified. A particular hybridoma can then be used for future, unlimited production of a highly specific antibody.
The availability of monoclonal antibodies has opened completely new possibilities for basic as well as applied biomedical research. The following examples of the use of monoclonal antibodies can be given.
1. Detailed studies of the distribution of different functions in different parts of antigen molecules. These studies may concern building elements of infectious agents; cell products such as enzymes and hormones; surface structures of cells etc. The mapping of variations in the surface components of influenza virus which explain the occurrence of repeated infections is one example.
2. High degree purification of substances, e.g. interferon, by taking advantage of the unique capacity displayed by a particular monoclonal antibody to bind to a certain antigen. In this case one uses a technique referred to as affinity chromatography.
3. Diagnostic characterization of diseases by identification of special structures on the surface or on the inside of cells. Hereby it is possible to distinguish between different forms of tumours and follow the development of tumours. Furthermore, it is possible to distinguish between different kinds of normal white blood cells. This is of importance for the characterization of certain immune deficiency conditions as seen e.g. in connection with the disease AIDS (acquired immune deficiency syndrome).Diseases caused by infectious agents can also be diagnosed by use of monoclonal antibodies. Thus, virus infected cells and bacteria or parasites inside or outside cells can be identified with a unique degree of specificity.
4. Treatment of AIDS diseases. Monoclonal antibodies against specialized white blood cells have been used with some success in connection with transplantation. There may also be possibilities to use monoclonal antibodies for treatment of tumours.

Recombinant DNA Technology in the Synthesis of Human Insulin

Recombinant DNA Technology in the Synthesis of Human Insulin
Since Banting and Best discovered the hormone, insulin in 1921. diabetic patients, whose elevated sugar levels are due to impaired insulin production, have been treated with insulin derived from the pancreas glands of abattoir animals. The hormone, produced and secreted by the beta cells of the pancreas' islets of Langerhans, regulates the use and storage of food, particularly carbohydrates.
Although bovine and porcine insulin are similar to human insulin, their composition is slightly different.Consequently, a number of patients' immune systems produce antibodies against it, thus neutralising its actions and resulting in inflammatory responses at injection sites. Added to these adverse effects of bovine and porcine insulin, were fears of long term complications ensuing from the regular injection of a foreign substance, as well as a projected decline in the production of animal derived insulin.These factors led researchers to consider synthesising Humulin by inserting the insulin gene into a suitable vector, the E. coli bacterial cell, to produce an insulin that is chemically identical to its naturally produced counterpart.
This has been achieved using Recombinant DNA technology.
This method is a more reliable and sustainable method than extracting and purifying the abattoir by-product.Understanding the genetics involved.
The structure of insulin.
Chemically, insulin is a small, simple protein. It consists of 51 amino acid, 30 of which constitute one polypeptide chain, and 21 of which comprise a second chain. The two chains are linked by a disulfide bond.
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Inside the Double Helix.
The genetic code for insulin is found in the DNA at the top of the short arm of the eleventh chromosome. It contains 153 nitrogen bases (63 in the A chain and 90 in the B chain).DNA Deoxyribolnucleic Acid), which makes up the chromosome, consists of two long intertwined helices, constructed from a chain of nucleotides, each composed of a sugar deoxyribose, a phosphate and nitrogen base. There are four different nitrogen bases, adenine, thymine, cytosine and guanine.The synthesis of a particular protein such as insulin is determined by the sequence in which these bases
are repeated .
Insulin synthesis from the genetic code.
The double strand of the eleventh chromosome of DNA divides in two, exposing unpaired nitrogen bases which are specific to insulin production .
The role of the mRNA strand, on which the nitrogen base thymine is replaced by uracil, is to carry genetic information, such as that pertaining to insulin,from the nucleus into the cytoplasm, where it attaches to a ribosome
The nitrogen bases on the mRNA are grouped into threes, known as codons. Transfer RNA (tRNA) molecules, three unpaired nitrogen bases bound to a specific amino acid, collectively known as an anti-codon pair with complementary bases (the codons) on the mRNA. The Vector (Gram negative E. coli).
A weakened strain of the common bacterium, Escherrichia coli (E. coli), an inhabitant of the human digestive tract, is the 'factory' used in the genetic engineering of insulin. When the bacterium reproduces, the insulin gene is replicated along with the plasmid, a circular section of DNA . E. coli produces enzymes that rapidly degrade foreign proteins such as insulin. By using mutant strains that lack these enzymes, the problem is avoidedIn E. coli, B-galactosidase is the enzyme that controls the transcription of the genes. To make the bacteria produce insulin, the insulin gene needs to be tied to this enzyme. Inside the genetic engineer's toolbox
Restriction enzymes, naturally produced by bacteria, act like biological scalpels, only recognising particular stretches of nucleotides, such as the one that codes for insulin.
This makes it possible to sever certain nitrogen base pairs and remove the section of insulin coding DNA from one organism's chromosome so that it can manufacture insulin . DNA ligase is an enzyme which serves as a genetic glue, welding the sticky ends of exposed nucleotides together.
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Manufacturing Human insulin.

The first step is to chemically synthesise the DNA chains that carry the specific nucleotide sequences characterising the A and B polypeptide chains of insulin The required DNA sequence can be determined because the amino acid compositions of both chains have been charted. Sixty three nucleotides are required for synthesising the A chain and ninety for the B chain, plus a codon at the end of each chain,signalling the termination of protein synthesis. An anti-codon, incorporating the amino acid, methionine, is then placed at the beginning of each chain which allows the removal of the insulin protein from the bacterial cell's amino acids.
The synthetic A and B chain 'genes' are then separately inserted into the gene for a bacterial enzyme, B-galactosidase, which is carried in the vector's plasmid. At this stage, it is crucial to ensure that the codons of the synthetic gene are compatible with those of the B-galactosidase. The recombinant plasmids are then introduced into E. coli cells. Practical use of Recombinant DNA technology in the synthesis of human insulin requires millions of copies of the bacteria whose plasmid
has been combined with the insulin gene in order to yield insulin. The insulin gene is expressed as it replicates with the B-galactosidase in the cell undergoing mitosisThe protein which is formed, consists partly of B-galactosidase, joined to either the A or B chain of insulin . The A and B chains are then extracted from the B-galactosidase fragment and purified. Biological implications of genetically engineered Recombinant human insulin. Human insulin is the only animal protein to have been made in bacteria in such a way that its structure is absolutely identical to that of the natural molecule. This reduces the possibility of complications resulting from antibody production. In chemical and pharmacological studies, commercially available Recombinant DNA human insulin has proven indistinguishable from pancreatic human insulin. Initially the major difficulty encountered was the contamination of the final product by the host cells, increasing the risk of contamination in the fermentation broth. This danger was eradicated by the introduction of purification processes. When the final insulin product is subjected to a battery of tests, including the finest radio-immuno assay techniques, no impurities can be detected. The entire procedure is now performed using yeast cells as a growth medium, as they secrete an almost complete human insulin molecule with perfect three dimensional structure.
This minimises the need for complex and costly purification procedures. The issue of hypoglycaemic complications in the administration of human insulin.
Since porcine insulin was phased out, and the majority of insulin dependent patients are now treated with genetically engineered recombinant human insulin, doctors and patients have become concerned about the increase in the number of hypoglycaemic episodes experienced. Although hypoglycaemia can be expected occasionally with any type of insulin, some people with diabetes claim that they are less cognisant of attacks of hypoglycaemia since switching from animal derived insulin to Recombinant DNA human insulin.(16) In a British study,
published in the 'Lancet", hypoglycaemia was induced in patients using either pork or human insulin, The researchers found "no significant difference in the frequency of signs of hypoglycaemia between users of the two different types of insulin."
An anecdotal report from a British patient who had been insulin dependent for thirty years, stated that she began experiencing recurring, unheralded hypoglycaemia only after substituting Recombinant DNA human insulin for animal derived insulin. After switching back to pork insulin to ease her mind, she hadn't experienced any unannounced hypoglycaemia. Eli Lilly and Co., a manufacturer of human insulin, noted that a third of people with diabetes, who have been insulin dependent for over ten years, "lose their hypoglycaemic warning signals, regardless of the type of insulin they are taking." Dr Simon P. Wolff of the University College of London said in an issue of Nature , "As far as I can make out, there's no fault (with the human insulin)." He concluded, "I do think we need to have a study to examine the possible risk.

Sterile filtration

Sterile filtration

Clear liquids that would be damaged by heat, irradiation or chemical sterilization can be sterilized by mechanical filtration. This method is commonly used for sensitive pharmaceuticals and protein solutions in biological research. A filter with pore size 0.2 µm will effectively remove bacteria. If viruses must also be removed, a much smaller pore size around 20 nm is needed. Solutions filter slowly through membranes with smaller pore diameters. Prions are not removed by filtration. The filtration equipment and the filters themselves may be purchased as presterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure sterility, the filtration system must be tested to ensure that the membranes have not been punctured prior to or during use.
To ensure the best results, pharmaceutical sterile filtration is performed in a room with highly filtered air (HEPA filter) or in a laminar flow cabinet or "flowbox", a device which produces a laminar stream of HEPA filtered air.

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Radiation sterilization

Radiation sterilization
Methods exist to sterilize using radiation such as electron beams, X-rays, gamma rays, or subatomic particles.
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Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, cannulas and IV sets. Gamma radiation requires bulky shielding for the safety of the operators; they also require storage of a radioisotope (usually Cobalt-60), which continuously emits gamma rays (it cannot be turned off, and therefore always presents a hazard in the area of the facility). Electron beam processing is also commonly used for medical device sterilization. Electron beams use an on-off technology and provide a much higher dosing rate then gamma or x-rays. Due to the higher dose rate, less exposure time is needed and thereby any potential degradation to polymers is reduced. A limitation is that electron beams are less penetrating than either gamma or x-rays. X-rays are less penetrating than gamma rays and tend to require longer exposure times, but require less shielding, and are generated by an X-ray machine that can be turned off for servicing and when not in use. Ultraviolet light irradiation (UV, from a germicidal lamp) is useful only for sterilization of surfaces and some transparent objects. Many objects that are transparent to visible light absorb UV. UV irradiation is routinely used to sterilize the interiors of biological safety cabinets between uses, but is ineffective in shaded areas, including areas under dirt (which may become polymerized after prolonged irradiation, so that it is very difficult to remove). It also damages many plastics, such as polystyrene foam. Further information: Ultraviolet Germicidal Irradiation Subatomic particles may be more or less penetrating, and may be generated by a radioisotope or a device, depending upon the type of particle. Irradiation with X-rays or gamma rays does not make materials radioactive. Irradiation with particles may make materials radioactive, depending upon the type of particles and their energy, and the type of target material: neutrons and very high-energy particles can make materials radioactive, but have good penetration, whereas lower energy particles (other than neutrons) cannot make materials radioactive, but have poorer penetration.
Irradiation is used by the United States Postal Service to sterilize mail in the Washington, DC area. Some foods (e.g. spices, ground meats) are irradiated for sterilization (see food irradiation).

Chemical sterilization

Chemical sterilization
Chemicals are also used for sterilization. Although heating provides the most reliable way to rid objects of all transmissible agents, it is not always appropriate, because it will damage heat-sensitive materials such as biological materials, fiber optics, electronics, and many plastics.
Ethylene oxide (EO or EtO) gas is commonly used to sterilize objects sensitive to temperatures greater than 60 °C such as plastics, optics and electrics. Ethylene oxide treatment is generally carried out between 30 °C and 60 °C with relative humidity above 30% and a gas concentration between 200 and 800 mg/L for at least three hours. Ethylene oxide penetrates well, moving through paper, cloth, and some plastic films and is highly effective. Ethylene oxide sterilizers are used to process sensitive instruments which cannot be adequately sterilized by other methods. EtO can kill all known viruses, bacteria and fungi, including bacterial spores and is satisfactory for most medical materials, even with repeated use. However it is highly flammable, and requires a longer time to sterilize than any heat treatment. The process also requires a period of post-sterilization aeration to remove toxic residues. Ethylene oxide is the most common sterilization method, used for over 70% of total sterilizations, and for 50% of all disposable medical devices.
The two most important ethylene oxide sterilization methods are: (1) the gas chamber method and (2) the micro-dose method. To benefit from economies of scale, EtO has traditionally been delivered by flooding a large chamber with a combination of EtO and other gases used as dilutants (usually CFCs or carbon dioxide ). This method has drawbacks inherent to the use of large amounts of sterilant being released into a large space, including air contamination produced by CFCs and/or large amounts of EtO residuals, flammability and storage issues calling for special handling and storage, operator exposure risk and training costs. Because of these problems a micro-dose sterilization method was developed in the late 1950s, using a specially designed bag to eliminate the need to flood a larger chamber with EtO. This method is also known as gas diffusion sterilization, or bag sterilization. This method minimize the use of gas.[4]
Bacillus subtilis, a very resistant organism, is used as a rapid biological indicator for EO sterilizers. If sterilization fails, incubation at 37 °C causes a fluorescent change within four hours, which is read by an auto-reader. After 96 hours, a visible color change occurs. Fluorescence is emitted if a particular (EO resistant) enzyme is present, which means that spores are still active. The color change indicates a pH shift due to bacterial metabolism. The rapid results mean that the objects treated can be quarantined until the test results are available.
Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for surfaces. It has the benefit of being able to oxidize most organic matter. On the other hand, it is a toxic and unstable gas that must be produced on-site, so it is not practical to use in many settings.
Chlorine bleach is another accepted liquid sterilizing agent. Household bleach consists of 5.25% sodium hypochlorite. It is usually diluted to 1/10 immediately before use; however to kill Mycobacterium tuberculosis it should be diluted only 1/5. The dilution factor must take into account the volume of any liquid waste that it is being used to sterilize.[5] Bleach will kill many organisms immediately, but for full sterilization it should be allowed to react for 20 minutes. Bleach will kill many, but not all spores. It is highly corrosive and may corrode even stainless steel surgical instruments.
Glutaraldehyde and formaldehyde solutions (also used as fixatives) are accepted liquid sterilizing agents, provided that the immersion time is sufficiently long. To kill all spores in a clear liquid can take up to 12 hours with glutaraldehyde and even longer with formaldehyde. The presence of solid particles may lengthen the required period or render the treatment ineffective. Sterilization of blocks of tissue can take much longer, due to the time required for the fixative to penetrate. Glutaraldehyde and formaldehyde are volatile, and toxic by both skin contact and inhalation. Glutaraldehyde has a short shelf life (<2>Ortho-phthalaldehyde (OPA) is a chemical sterilizing agent that received Food and Drug Administration (FDA) clearance in late 1999. Typically used in a 0.55% solution, OPA shows better myco-bactericidal activity than glutaraldehyde. It also is effective against glutaraldehyde-resistant spores. OPA has superior stability, is less volatile, and does not irritate skin or eyes, and it acts more quickly than glutaraldehyde. On the other hand, it is more expensive, and will stain proteins (including skin) gray in color.
Hydrogen peroxide is another chemical sterilizing agent. It is relatively non-toxic once diluted to low concentrations (although a dangerous oxidizer at high concentrations), and leaves no residue.
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Low Temperature Plasma sterilization chambers use hydrogen peroxide vapor to sterilize heat-sensitive equipment such as rigid endoscopes. A recent model can sterilize most hospital loads in as little as 20 minutes. The Sterrad has limitations with processing certain materials such as paper/linens and long thin lumens. Paper products cannot be sterilized in the Sterrad system because of a process called cellulostics, in which the hydrogen peroxide would be completely absorbed by the paper product.
Hydrogen peroxide and formic acid are mixed as needed in the Endoclens device for sterilization of endoscopes. This device has two independent asynchronous bays, and cleans (in warm detergent with pulsed air), sterilizes and dries endoscopes automatically in 30 minutes. Studies with synthetic soil with bacterial spores showed the effectiveness of this device.
Dry sterilization process (DSP) uses hydrogen peroxide at a concentration of 30-35% under low pressure conditions. This process achieves bacterial reduction of 10-6...10-8. The complete process cycle time is just 6 seconds, and the surface temperature is increased only 10-15 °C (18 to 27 °F). Originally designed for the sterilization of plastic bottles in the beverage industry, because of the high germ reduction and the slight temperature increase the dry sterilization process is also useful for medical and pharmaceutical applications.
Peracetic acid (0.2%) is used to sterilize instruments in the Steris system.
Prions are highly resistant to chemical sterilization. Treatment with aldehydes (e.g., formaldehyde) have actually been shown to increase prion resistance. Hydrogen peroxide (3%) for one hour was shown to be ineffective, providing less than 3 logs (10-3) reduction in contamination. Iodine, formaldehyde, glutaraldehyde and peracetic acid also fail this test (one hour treatment). Only chlorine, a phenolic compound, guanidinium thiocyanate, and sodium hydroxide (NaOH) reduce prion levels by more than 4 logs. Chlorine and NaOH are the most consistent agents for prions. Chlorine is too corrosive to use on certain objects. Sodium hydroxide has had many studies showing its effectiveness.
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Other methods of sterilisation

Other methods of sterilisation
Other heat methods include flaming, incineration, boiling, tindalization, and using dry heat.
Flaming is done to loops and straight-wires in microbiology labs. Leaving the loop in the flame of a Bunsen burner or alcohol lamp until it glows red ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating infectious material may be "sprayed" from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area.
Incineration will also burn any b organism to ash. It is used to sanitize medical and other biohazardous waste before it is discarded with non-hazardous waste.
Boiling in water for fifteen minutes will kill most vegetative bacteria and viruses, but boiling is ineffective against prions and many bacterial and fungal spores; therefore boiling is unsuitable for sterilization. However, since boiling does kill most vegetative microbes and viruses, it is useful for reducing viable levels if no better method is available. Boiling is a simple process, and is an option available to most people, requiring only water, enough heat, and a container that can withstand the heat; however, boiling can be hazardous and cumbersome.
Tindalization[2] /Tyndallization[3] named after John Tyndall is a lengthy process designed to reduce the level of activity of sporulating bacteria that are left by a simple boiling water method. The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling, cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again. The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. This is effective because many spores are stimulated to grow by the heat shock. The procedure only works for media that can support bacterial growth - it will not sterilize plain water. Tindalization/tyndallization is ineffective against prions.
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Dry heat can be used to sterilize items, but as the heat takes much longer to be transferred to the organism, both the time and the temperature must usually be increased, unless forced ventilation of the hot air is used. The standard setting for a hot air oven is at least two hours at 160 °C (320 °F). A rapid method heats air to 190 °C (374 °F) for 6 minutes for unwrapped objects and 12 minutes for wrapped objects.[1][2] Dry heat has the advantage that it can be used on powders and other heat-stable items that are adversely affected by steam (for instance, it does not cause rusting of steel objects). Prions can be inactivated by immersion in sodium hydroxide (NaOH 0.09N) for two hours plus one hour autoclaving (121 °C/250 °F). Several investigators have shown complete (>7.4 logs) inactivation with this combined treatment. However, sodium hydroxide may corrode surgical instruments, especially at the elevated temperatures of the autoclave.
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STERILISATION METHODS

Sterilization
Sterilization (or sterilisation, see spelling differences) refers to any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses, prions and spore forms etc.) from a surface, equipment, foods, medications, or biological culture medium. Sterilization can be achieved through application of heat, chemicals, irradiation, high pressure or filtration.ClassificationThere are two types of sterilization: physical and chemical.
Physical sterilization includes:
heat sterilization radiation sterilization Chemical sterilization includes:
ethylene oxide ozone chlorine bleach glutaraldehyde formaldehyde hydrogen peroxide peracetic acidv prions
ApplicationsFoodsThe first application of sterilization was thorough cooking to effect the partial heat sterilization of foods and water. Cultures that practice heat sterilization of food and water have longer life expectancy and lower rates of disability. Canning of foods by heat sterilization was an extension of the same principle. Ingestion of contaminated food and water remains a leading cause of illness and death in the developing world, particularly for children.
Medicine and surgeryIn general, surgical instruments and medications that enter an already sterile part of the body (such as the blood, or beneath the skin) must have a high sterility assurance level. Examples of such instruments include scalpels, hypodermic needles and artificial pacemakers. This is also essential in the manufacture of parenteral pharmaceuticals.
Heat sterilization of medical instruments is known to have been used in Ancient Rome, but it mostly disappeared throughout the Middle Ages resulting in significant increases in disability and death following surgical procedures.
Preparation of injectable medications and intravenous solutions for fluid replacement therapy requires not only a high sterility assurance level, but well-designed containers to prevent entry of adventitious agents after initial sterilization.
Food
sterilization is usually considered a harsher form of Pasteurization[1], and is carried out through heating, though other methods are available. Food sterilization is commonly a part of canning and is used in combination with or instead of preservatives, refrigeration, and other ways to preserve food.
Heat sterilizationDry heat sterilization Moist heat sterilization
Steam sterilization
A widely-used method for heat sterilization is the autoclave. Autoclaves commonly use steam heated to 121 °C or 134 °C. To achieve sterility, a holding time of at least 15 minutes at 121 °C or 3 minutes at 134 °C is required. Additional sterilizing time is usually required for liquids and instruments packed in layers of cloth, as they may take longer to reach the required temperature. After sterilization, autoclaved liquids must be cooled slowly to avoid boiling over when the pressure is released.For prion elimination, various recommendations state 121–132 °C (270 °F) for 60 minutes or 134 °C (273 °F) for at least 18 minutes. The prion that causes the disease scrapie (strain 263K) is inactivated relatively quickly by such sterilization procedures; however, other strains of scrapie, as well as strains of CJD and BSE are more resistant. Using mice as test animals, one experiment showed that heating BSE positive brain tissue at 134-138 °C (273-280 °F) for 18 minutes resulted in only a 2.5 log decrease in prion infectivity. (The initial BSE concentration in the tissue was relatively low). For a significant margin of safety, cleaning should reduce infectivity by 4 logs, and the sterilization method should reduce it a further 5 logs.
To ensure the autoclaving process was able to cause sterilization, most autoclaves have meters and charts that record or display pertinent information such as temperature and pressure as a function of time. Indicator tape is often placed on packages of products prior to autoclaving. A chemical in the tape will change color when the appropriate conditions have been met. Some types of packaging have built-in indicators on them.
Biological indicators ("bioindicators") can also be used to independently confirm autoclave performance. Simple bioindicator devices are commercially available based on microbial spores. Most contain spores of the heat resistant microbe Bacillus stearothermophilus, among the toughest organisms for an autoclave to destroy. Typically these devices have a self-contained liquid growth medium and a growth indicator. After autoclaving an internal glass ampule is shattered, releasing the spores into the growth medium. The vial is then incubated (typically at 56 °C (132 °F)) for 48 hours. If the autoclave destroyed the spores, the medium will remain its original color. If autoclaving was unsuccessful the B. sterothermophilus will metabolize during incubation, causing a color change during the incubation.
For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave must not be overcrowded, and the lids of bottles and containers must be left ajar. During the initial heating of the chamber, residual air must be removed. Indicators should be placed in the most difficult places for the steam to reach to ensure that steam actually penetrates there.
For autoclaving, as for all disinfection of sterilization methods, cleaning is critical. Extraneous biological matter or grime may shield organisms from the property intended to kill them, whether it physical or chemical. Cleaning can also remove a large number of organisms. Proper cleaning can be achieved by physical scrubbing. This should be done with detergent and warm water to get the best results. Cleaning instruments or utensils with organic matter, cool water must be used because warm or hot water may cause organic debris to coagulate. Treatment with ultrasound or pulsed air can also be used to remove debris.
FoodAlthough imperfect, cooking and canning are the most common applications of heat sterilization. Boiling water kills the vegetative stage of all common microbes. Roasting meat until it is well done typically completely sterilizes the surface. Since the surface is also the part of food most likely to be contaminated by microbes, roasting usually prevents food poisoning. Note that the common methods of cooking food do not sterilize food - they simply reduce the number of disease-causing micro-organisms to a level that is not dangerous for people with normal digestive and immune systems.
Pressure cooking is analogous to autoclaving and when performed correctly renders food sterile. However, some foods are notoriously difficult to sterilize with home canning equipment, so expert recommendations should be followed for home processing to avoid food poisoning.
Food utensils
Dishwashers often only use hot tap water or heat the water to between 49 and 60 °C (120 and 140 °F), and thus provide temperatures that could promote bacterial growth. That is to say, they do not effectively sterilize utensils. Some dishwashers do actually heat water up to 74 °C (165 °F) or higher; those often are specifically described as having sterilization modes of some sort, but this is not a substitute for autoclaving.
Note that dishwashers remove food traces from the utensils by a combination of mechanical action (the action of water hitting the plates and cutlery) and the action of detergents and enzymes on fats and proteins. This removal of food particles thus removes one of the factors required for bacterial growth (food), it clearly explains why items with cracks and crevices should either be washed by hand or disposed of: if the water cannot get to the area needing cleaning, the warm, moist, dark conditions in the dishwasher can actually promote bacterial growth.
Bathing
Bathing and washing are not hot enough to sterilize bacteria without scalding the skin. Most hot tap water is between 43 and 49 °C (110 and 120 °F), though some people set theirs as high as 55 °C (130 °F). Humans begin to find water painful at 41 to 42 °C (106 to 108 °F), which to many bacteria is just starting to get warm enough for them to grow quickly; they will grow faster, rather than be killed at temperatures up to 55 °C (130 °F) or more.

Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions.

Cleanroom

Cleanroom
A cleanroom is an environment, typically used in manufacturing or scientific research, that has a low level of environmental pollutants such as dust, airborne microbes, aerosol particles and chemical vapors. More accurately, a cleanroom has a controlled level of contamination that is specified by the number of particles per cubic meter at a specified particle size. To give a perspective, the ambient air outside in a typical urban environment might contain as many as 35,000,000 particles per cubic meter, 0.5 μm and larger in diameter, corresponding to an ISO class 9 cleanroom.
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Cleanrooms can be very large. Entire manufacturing facilities can be contained within a cleanroom with factory floors covering thousands of square meters. They are used extensively in semiconductor manufacturing, biotechnology, the life sciences and other fields that are very sensitive to environmental contamination.
The air entering a cleanroom from outside is filtered to exclude dust, and the air inside is constantly recirculated through high efficiency particulate air (HEPA) and ultra low penetration air (ULPA) filters to remove internally generated contaminants.
Staff enter and leave through airlocks (sometimes including an air shower stage), and wear protective clothing such as hats, face masks, gloves, boots and cover-alls.
Equipment inside the cleanroom is designed to generate minimal air contamination. There are even specialised mops and buckets. Cleanroom furniture is also designed to produce a low amount of particles and to be easy to clean.
Common materials such as paper, pencils, and fabrics made from natural fibers are often excluded; however, alternatives are available. Cleanrooms are not sterile (i.e., free of uncontrolled microbes) [1]and more attention is given to airborne particles. Particle levels are usually tested using a particle counter.
Some cleanrooms are kept at a positive pressure so that if there are any leaks, air leaks out of the chamber instead of unfiltered air coming in.
Some cleanroom HVAC systems control the humidity to relatively low levels, such that extra precautions are necessary to prevent ESD electrostatic discharge problems. These ESD controls ("ionizers") are also used in rooms where ESD sensitive products are produced or handled.
Low-level cleanrooms may only require special shoes, ones with completely smooth soles that do not track in dust or dirt. However, shoe bottoms must not create slipping hazards (safety always takes precedence). Entering a cleanroom usually requires wearing a cleanroom suit.
In cheaper cleanrooms, in which the standards of air contamination are less rigorous, the entrance to the cleanroom may not have an air shower. There is an anteroom, in which the special suits must be put on, but then a person can walk in directly to the room (as seen in the photograph on the right).
Some manufacturing facilities do not use fully classified cleanrooms, but use some cleanroom practices together to maintain their cleanliness requirements.
Clean room classifications
Clean rooms are classified according to the number and size of particles permitted per volume of air. Large numbers like "class 100" or "class 1000" refer to US FED STD 209E, and denote the number of particles of size 0.5 µm or larger permitted per cubic foot of air. The standard also allows interpolation, so it is possible to describe e.g. "class 2000".
Small numbers refer to ISO 14644-1 standards, which specify the decimal logarithm of the number of particles 0.1 µm or larger permitted per cubic metre of air. So, for example, an ISO class 5 clean room has at most 105 = 100,000 particles per m³.
Note that both FS 209E and ISO 14644-1 are based on assumed log-log relationships between particle size and particle concentration. For that reason, there are no "zero" particle concentrations listed. The table locations without entries are N/A ("not applicable") combinations of particle sizes and cleanliness classes. They should not be read as zero.
Because 1 m³ is approximately 35 ft³, the two standards are mostly equivalent when measuring 0.5 µm particles, although the testing standards differ. Ordinary room air is approximately class 1,000,000 or ISO 9.US FED STD 209E cleanroom standards
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Types of contamination

Types of contaminationThere are many types of organism that are potentially detrimental to processes in a critical environment. Seven of the most common contaminants are:
Aspergillus niger
Clostridium difficile
Escherichia coli
Methicillin Resistant Staphylococcus aureus (MRSA)
Pseudomonas aeruginosa
Pseudomonas cepacia
Salmonella enteritidis
These, and many other damaging contaminants can infiltrate critical areas in a manner of ways. Particulate can enter by air, foot, or on any carrier between the external environment and inside the critical area.
The effects of contamination.
Contamination poses a significant risk to businesses as well as the individual. Unguarded proliferation of contamination can quickly lead to product damage, yield reduction, product recalls and other outcomes highly detrimental to businesses. A number of products over a range of industries are recalled due to ineffective contamination control systems.
By this evidence it could be argued that many businesses are not adequately protecting themselves from the harmful effects of contamination, and many products over many industries are being recalled due to unsafe manufacturing processes.
Is this really an acceptable attitude and policy from one of the government agencies responsible for the safety of the nation’s food supply? Where is the public outrage and demand for accountability? Perhaps the pointless prattling of politicians and of the television media’s “blabberatzi” has simply immunised us all from any reaction to the flagrant government agency unresponsiveness and blatant incompetence. It must be so, since years and years of recalls and years and years of studies, renewed attention, promises, and platitudes have left us with the same resultsTypes of contamination controlBody movement causes contamination and protective clothing such as hats, cleanroom suits and face masks are basic forms of contamination control. Apart from people, the other common way for contamination to enter is on the wheels of trolleys used to transport equipment.
To prevent airborne contamination, high efficiency particulate air (HEPA) filters, airlocks and cleanroom suits are used. HEPA filtration systems used in the medical sector incorporate high-energy ultra-violet light units to kill off the live bacteria and viruses trapped by the filter media. These measures restrict the number of particulates within the atmosphere, and inhibit growth in those that are viable.
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Studies by 3M show that over 80% of contamination enters the cleanroom through entrances and exits, mostly at or near floor level. To combat this suitable flooring systems are used that effectively attract, retain and inhibit growth of viable organisms. Studies show that the most effective type of flooring system is one of polymer composition.
Polymer mats are particularly effective due to their suppleness as they allow for more contact with serration on shoes and wheels and can accommodate for more particles whilst remaining effective. An electrostatic potential adds to the effectiveness of this type of contamination control as it holds particles until being cleaned. This method of attracting and retaining particles is more effective than mats with an active adhesive coating which needs to be peeled and is often not as supple. As long as the tack level of the mat is greater than the donor (foot or wheel), the contamination touching the surface will be removed. Very high tack surfaces pose a contamination threat because they are prone to pulling off over-shoe protection.Polymeric flooring is produced to ensure a higher level of tackiness than the surfaces it comes into contact with, without causing discomfort and potentially damaging ‘stickiness’
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Where is contamination control used?

Contamination controlis the collective name for any method that effectively controls the growth and proliferation of contamination. Contamination control may refer both to contamination prevention as well as to decontamination (i.e. controlling the spread of contamination from a hazardous materials site, etc.).FunctionContamination control is one of the most vital aspects of health and safety in areas where environmental sterility is a critical concern. The purpose of the wide range of contamination control procedures and standards is to ensure cleanliness by reducing or eradicating all viable and non-viable contamination, and maintaining an efficient rate of production.
Where is contamination control used?One of the most common environments that incorporates contamination control into its standards protocol is the cleanroom. There are many preventative procedures in place within the cleanroom environment. Procedures include subjecting cleanroom staff to strict clothing regulations, and there is often a gowning room where the staff can change under sterile conditions, so as to prevent any particulate from entering from the outside environment. Certain areas in the cleanroom have more stringent measures than others. Places such as packaging areas, corridors, gowning rooms and transfer hatches incorporate strict contamination control measures in order to keep to the cleanroom standards.
Contamination control is also an important asset to laboratories in industries such as the pharmaceutical and life science sectors. Other places of use include automotive paint shops, entrances to industrial kitchens and food service providers, many manufacturing areas, and in electronic component assembly areas.
More recently contamination control has been a concern for laboratories and other sensitive environments as an effective bio-security crisis management measure. Some banks and insurance companies use contamination control products as part of their disaster management protocols. Preventative measures are put in place as preparation for potential pandemics or the proliferation of biohazards in any potential terrorist attack.

Human microbial flora

Human flora

The human flora is the microrganisms that constantly inhabit the human body. They include bacteria, fungi and archaea. Some of these organisms are known to perform tasks that are useful for the human host, while the majority have no beneficial or harmful effect. Those that are expected to be present, and that under normal circumstances do not cause disease, are termed normal flora,or microbiota. An effort to better describe the microflora of humans has been initiated; see Human microbiome project.
Bacterial flora
It is estimated that 500 to 100,000 species of bacteria live in the human body . Bacterial cells are much smaller than human cells, and there are about ten times as many bacteria as human cells in the body (1000 trillion or 1 quadrillion (1015) versus 100 trillion (1014)).[2] Though normal flora are found on all surfaces exposed to the environment (on the skin and eyes, in the mouth, nose, small intestine, and colon), the vast majority of bacteria live in the large intestine.
Many of the bacteria in the digestive tract, collectively referred to as gut flora, are able to break down certain nutrients such as carbohydrates that humans otherwise could not digest. The majority of these commensal bacteria are anaerobes, meaning they survive in an environment with no oxygen. Bacteria of the normal flora can act as opportunistic pathogens at times of lowered immunity.
Escherichia coli (a.k.a. E. coli) is a bacterium that lives in the colon; it is an extensively studied model organism and probably the best understood bacterium of all Certain mutated strains of these gut bacteria do cause disease; an example is E. coli O157:H7.
A number of types of bacteria, such as Actinomyces viscosus and A. naeslundii, live in the mouth, where they are part of a sticky substance called plaque. If this is not removed by brushing, it hardens into calculus (also called tartar). The same bacteria also secrete acids that dissolve tooth enamel, causing tooth decay.
The vaginal microflora consist mostly of various lactobacillus species. It was long thought that the most common of these species was Lactobacillus acidophilus, but it has later been shown that the most common one is L. iners followed by L. crispatus. Other lactobacilli found in the vagina are L. delbruekii and L. gasseri. Disturbance of the vaginal flora can lead to bacterial vaginosis.
Human bacterial flora and human health
Bacteria are vital for the maintenance of human health, but some pathogenic bacteria also pose a significant health threat by causing diseases. Large numbers of bacteria live on the skin and in the digestive tract. Their growth can be increased by warmth and sweat. Large populations of these organisms on humans are the cause of body odor and thought to play a part in acne. There are more than 500 bacterial species present in the normal human gut and are generally beneficial: they synthesize vitamins such as folic acid, vitamin K and biotin, and they ferment complex indigestible carbohydrates. Other beneficial bacteria in the normal flora include Lactobacillus species, which convert lactose and other sugars to lactic acid in the gut.[6] The presence of such bacterial colonies also inhibits the growth of potentially pathogenic bacteria (usually through competitive exclusion) and some beneficial bacteria are consequently sold as probiotic dietary supplements.Archaea are present in the human gut, but in contrast to the enormous variety of bacteria in this organ, the number of archaeal species are much more limited.The dominant group is the methanogens, particularly Methanobrevibacter smithii and Methanosphaera stadtmanae. However, colonization by methanogens is variable and only about 50% of humans have easily-detectable populations or these organisms.
Archaea and human healthAs of 2007, no clear examples of archaeal pathogens are known, although a relationship has been proposed between the presence of some methanogens and human periodontal disease.
Fungal floraFungi, particularly yeasts are present in the human gut. The best studied of these are Candida species, due to their ability to become pathogenic in immunocompromised hosts.[14] Yeasts are also present on the skin, particularly Malassezia species, where they consume oils secreted from the sebaceous glands.

Pathogens

If bacteria form a parasitic association with other organisms, they are classed as pathogens. Pathogenic bacteria are a major cause of human death and disease and cause infections such as tetanus, typhoid fever, diphtheria, syphilis, cholera, foodborne illness, leprosy and tuberculosis. A pathogenic cause for a known medical disease may only be discovered many years after, as was the case with Helicobacter pylori and peptic ulcer disease. Bacterial diseases are also important in agriculture, with bacteria causing leaf spot, fire blight and wilts in plants, as well as Johne's disease, mastitis, salmonella and anthrax in farm animals.
Each species of pathogen has a characteristic spectrum of interactions with its human hosts. Some organisms, such as Staphylococcus or Streptococcus, can cause skin infections, pneumonia, meningitis and even overwhelming sepsis, a systemic inflammatory response producing shock, massive vasodilation and death.Yet these organisms are also part of the normal human flora and usually exist on the skin or in the nose without causing any disease at all. Other organisms invariably cause disease in humans, such as the Rickettsia, which are obligate intracellular parasites able to grow and reproduce only within the cells of other organisms. One species of Rickettsia causes typhus, while another causes Rocky Mountain spotted fever. Chlamydia, another phylum of obligate intracellular parasites, contains species that can cause pneumonia, or urinary tract infection and may be involved in coronary heart disease.Finally, some species such as Pseudomonas aeruginosa, Burkholderia cenocepacia, and Mycobacterium avium are opportunistic pathogens and cause disease mainly in people suffering from immunosuppression or cystic fibrosis.
Bacterial infections may be treated with antibiotics, which are classified as bacteriocidal if they kill bacteria, or bacteriostatic if they just prevent bacterial growth. There are many types of antibiotics and each class inhibits a process that is different in the pathogen from that found in the host. An example of how antibiotics produce selective toxicity are chloramphenicol and puromycin, which inhibit the bacterial ribosome, but not the structurally different eukaryotic ribosome. Antibiotics are used both in treating human disease and in intensive farming to promote animal growth, where they may be contributing to the rapid development of antibiotic resistance in bacterial populations.Infections can be prevented by antiseptic measures such as sterilizating the skin prior to piercing it with the needle of a syringe, and by proper care of indwelling catheters. Surgical and dental instruments are also sterilized to prevent contamination and infection by bacteria. Disinfectants such as bleach are used to kill bacteria or other pathogens on surfaces to prevent contamination and further reduce the risk of infection.

Movement (Motility)

Movement
Motile bacteria can move using flagella, bacterial gliding, twitching motility or changes of buoyancy. In twitching motility, bacterial use their type IV pili as a grappling hook, repeatedly extending it, anchoring it and then retracting it with remarkable force .
Bacterial species differ in the number and arrangement of flagella on their surface; some have a single flagellum (monotrichous), a flagellum at each end (amphitrichous), clusters of flagella at the poles of the cell (lophotrichous), while others have flagella distributed over the entire surface of the cell (peritrichous). The bacterial flagella is the best-understood motility structure in any organism and is made of about 20 proteins, with approximately another 30 proteins required for its regulation and assembly. The flagellum is a rotating structure driven by a motor at the base that uses the electrochemical gradient across the membrane for power. This motor drives the motion of the filament, which acts as a propeller. Many bacteria (such as E. coli) have two distinct modes of movement: forward movement (swimming) and tumbling. The tumbling allows them to reorient and makes their movement a three-dimensional random walk. The flagella of a unique group of bacteria, the spirochaetes, are found between two membranes in the periplasmic space. They have a distinctive helical body that twists about as it moves.
Motile bacteria are attracted or repelled by certain stimuli in behaviors called taxes: these include chemotaxis, phototaxis and magnetotaxis. In one peculiar group, the myxobacteria, individual bacteria move together to form waves of cells that then differentiate to form fruiting bodies containing spores. The myxobacteria move only when on solid surfaces, unlike E. coli which is motile in liquid or solid media.
Several Listeria and Shigella species move inside host cells by usurping the cytoskeleton, which is normally used to move organelles inside the cell. By promoting actin polymerization at one pole of their cells, they can form a kind of tail that pushes them through the host cell's cytoplasm.

Genetics

Most bacteria have a single circular chromosome that can range in size from only 160,000 base pairs in the endosymbiotic bacteria Candidatus Carsonella ruddii, to 12,200,000 base pairs in the soil-dwelling bacteria Sorangium cellulosum. Spirochaetes of the genus Borrelia are a notable exception to this arrangement, with bacteria such as Borrelia burgdorferi, the cause of Lyme disease, containing a single linear chromosome. The genes in bacterial genomes are usually a single continuous stretch of DNA and although several different types of introns do exist in bacteria, these are much more rare than in eukaryotes. pharmaguideline.
Bacteria may also contain plasmids, which are small extra-chromosomal DNAs that may contain genes for antibiotic resistance or virulence factors. Another type of bacterial DNA are integrated viruses (bacteriophages). Many types of bacteriophage exist, some simply infect and lyse their host bacteria, while others insert into the bacterial chromosome. A bacteriophage can contain genes that contribute to its host's phenotype: for example, in the evolution of Escherichia coli O157:H7 and Clostridium botulinum, the toxin genes in an integrated phage converted a harmless ancestral bacteria into a lethal pathogen.
Bacteria, as asexual organisms, inherit identical copies of their parent's genes (i.e., they are clonal). However, all bacteria can evolve by selection on changes to their genetic material DNA caused by genetic recombination or mutations. Mutations come from errors made during the replication of DNA or from exposure to mutagens. Mutation rates vary widely among different species of bacteria and even among different clones of a single species of bacteria.Genetic changes in bacterial genomes come from either random mutation during replication or "stress-directed mutation", where genes involved in a particular growth-limiting process have an increased mutation rate. pharma .blogspot.com
Some bacteria also transfer genetic material between cells. This can occur in three main ways. Firstly, bacteria can take up exogenous DNA from their environment, in a process called transformation. Genes can also be transferred by the process of transduction, when the integration of a bacteriophage introduces foreign DNA into the chromosome. The third method of gene transfer is bacterial conjugation, where DNA is transferred through direct cell contact. This gene acquisition from other bacteria or the environment is called horizontal gene transfer and may be common under natural conditions. Gene transfer is particularly important in antibiotic resistance as it allows the rapid transfer of resistance genes between different pathogens.

Growth and Reproduction of Bacteria

Growth and reproduction
Unlike multicellular organisms, increases in the size of bacteria (cell growth) and their reproduction by cell division are tightly linked in unicellular organisms. Bacteria grow to a fixed size and then reproduce through binary fission, a form of asexual reproduction.[84] Under optimal conditions, bacteria can grow and divide extremely rapidly, and bacterial populations can double as quickly as every 9.8 minutes. In cell division, two identical clone daughter cells are produced. Some bacteria, while still reproducing asexually, form more complex reproductive structures that help disperse the newly-formed daughter cells. Examples include fruiting body formation by Myxobacteria and arial hyphae formation by Streptomyces, or budding. Budding involves a cell forming a protrusion that breaks away and produces a daughter cell.In the laboratory, bacteria are usually grown using solid or liquid media. Solid growth media such as agar plates are used to isolate pure cultures of a bacterial strain. However, liquid growth media are used when measurement of growth or large volumes of cells are required. Growth in stirred liquid media occurs as an even cell suspension, making the cultures easy to divide and transfer, although isolating single bacteria from liquid media is difficult. The use of selective media (media with specific nutrients added or deficient, or with antibiotics added) can help identify specific organisms.
Most laboratory techniques for growing bacteria use high levels of nutrients to produce large amounts of cells cheaply and quickly. However, in natural environments nutrients are limited, meaning that bacteria cannot continue to reproduce indefinitely. This nutrient limitation has led the evolution of different growth strategies (see r/K selection theory). Some organisms can grow extremely rapidly when nutrients become available, such as the formation of algal (and cyanobacterial) blooms that often occur in lakes during the summer. Other organisms have adaptations to harsh environments, such as the production of multiple antibiotics by Streptomyces that inhibit the growth of competing microorganisms. In nature, many organisms live in communities (e.g. biofilms) which may allow for increased supply of nutrients and protection from environmental stresses. These relationships can be essential for growth of a particular organism or group of organisms (syntrophy).
Bacterial growth follows three phases. When a population of bacteria first enter a high-nutrient environment that allows growth, the cells need to adapt to their new environment. The first phase of growth is the lag phase, a period of slow growth when the cells are adapting to the high-nutrient environment and preparing for fast growth. The lag phase has high biosynthesis rates, as proteins necessary for rapid growth are produced. The second phase of growth is the logarithmic phase (log phase), also known as the exponential phase. The log phase is marked by rapid exponential growth. The rate at which cells grow during this phase is known as the growth rate (k), and the time it takes the cells to double is known as the generation time (g). During log phase, nutrients are metabolised at maximum speed until one of the nutrients is depleted and starts limiting growth. The final phase of growth is the stationary phase and is caused by depleted nutrients. The cells reduce their metabolic activity and consume non-essential cellular proteins. The stationary phase is a transition from rapid growth to a stress response state and there is increased expression of genes involved in DNA repair, antioxidant metabolism and nutrient transport.

Bacterial Metabolism

Bacterial Metabolism
In contrast to higher organisms, bacteria exhibit an extremely wide variety of metabolic types. The distribution of metabolic traits within a group of bacteria has traditionally been used to define their taxonomy, but these traits often do not correspond with modern genetic classifications. Bacterial metabolism is classified on the basis of three major criteria: the kind of energy used for growth, the source of carbon, and the electron donors used for growth. An additional criterion of respiratory microorganisms are the electron acceptors used for aerobic or anaerobic respiration.
Carbon metabolism in bacteria is either heterotrophic, where organic carbon compounds are used as carbon sources, or autotrophic, meaning that cellular carbon is obtained by fixing carbon dioxide. Typical autotrophic bacteria are phototrophic cyanobacteria, green sulfur-bacteria and some purple bacteria, but also many chemolithotrophic species, such as nitrifying or sulfur-oxidising bacteria. Energy metabolism of bacteria is either based on phototrophy, the use of light through photosynthesis, or on chemotrophy, the use of chemical substances for energy, which are mostly oxidised at the expense of oxygen or alternative electron acceptors (aerobic/anaerobic respiration).
Finally, bacteria are further divided into lithotrophs that use inorganic electron donors and organotrophs that use organic compounds as electron donors. Chemotrophic organisms use the respective electron donors for energy conservation (by aerobic/anaerobic respiration or fermentation) and biosynthetic reactions (e.g. carbon dioxide fixation), whereas phototrophic organisms use them only for biosynthetic purposes. Respiratory organisms use chemical compounds as a source of energy by taking electrons from the reduced substrate and transferring them to a terminal electron acceptor in a redox reaction. This reaction releases energy that can be used to synthesise ATP and drive metabolism. In aerobic organisms, oxygen is used as the electron acceptor. In anaerobic organisms other inorganic compounds, such as nitrate, sulfate or carbon dioxide are used as electron acceptors. This leads to the ecologically important processes of denitrification, sulfate reduction and acetogenesis, respectively.
Another way of life of chemotrophs in the absence of possible electron acceptors is fermentation, where the electrons taken from the reduced substrates are transferred to oxidised intermediates to generate reduced fermentation products (e.g. lactate, ethanol, hydrogen, butyric acid). Fermentation is possible, because the energy content of the substrates is higher than that of the products, which allows the organisms to synthesise ATP and drive their metabolism.
These processes are also important in biological responses to pollution; for example, sulfate-reducing bacteria are largely responsible for the production of the highly toxic forms of mercury (methyl- and dimethylmercury) in the environment. Non-respiratory anaerobes use fermentation to generate energy and reducing power, secreting metabolic by-products (such as ethanol in brewing) as waste. Facultative anaerobes can switch between fermentation and different terminal electron acceptors depending on the environmental conditions in which they find themselves.
Lithotrophic bacteria can use inorganic compounds as a source of energy. Common inorganic electron donors are hydrogen, carbon monoxide, ammonia (leading to nitrification), ferrous iron and other reduced metal ions, and several reduced sulfur compounds. Unusually, the gas methane can be used by methanotrophic bacteria as both a source of electrons and a substrate for carbon anabolism. In both aerobic phototrophy and chemolithotrophy, oxygen is used as a terminal electron acceptor, while under anaerobic conditions inorganic compounds are used instead. Most lithotrophic organisms are autotrophic, whereas organotrophic organisms are heterotrophic.
In addition to fixing carbon dioxide in photosynthesis, some bacteria also fix nitrogen gas (nitrogen fixation) using the enzyme nitrogenase. This environmentally important trait can be found in bacteria of nearly all the metabolic types listed above, but is not universal.

Morphology of bacteria

Bacteria display a wide diversity of shapes and sizes, called morphologies. Bacterial cells are about one tenth the size of eukaryotic cells and are typically 0.5–5.0 micrometres in length. However, a few species–for example Thiomargarita namibiensis and Epulopiscium fishelsoni–are up to half a millimetre long and are visible to the unaided eye. Among the smallest bacteria are members of the genus Mycoplasma, which measure only 0.3 micrometres, as small as the largest viruses. pharma guideline
Most bacterial species are either spherical, called cocci (sing. coccus, from Greek kókkos, grain, seed) or rod-shaped, called bacilli (sing. bacillus, from Latin baculus, stick). Some rod-shaped bacteria, called vibrio, are slightly curved or comma-shaped; others, can be spiral-shaped, called spirilla, or tightly coiled, called spirochaetes. A small number of species even have tetrahedral or cuboidal shapes. This wide variety of shapes is determined by the bacterial cell wall and cytoskeleton, and is important because it can influence the ability of bacteria to acquire nutrients, attach to surfaces, swim through liquids and escape predators.
Many bacterial species exist simply as single cells, others associate in characteristic patterns: Neisseria form diploids (pairs), Streptococcus form chains, and Staphylococcus group together in "bunch of grapes" clusters. Bacteria can also be elongated to form filaments, for example the Actinobacteria. Filamentous bacteria are often surrounded by a sheath that contains many individual cells; certain types, such as species of the genus Nocardia, even form complex, branched filaments, similar in appearance to fungal mycelia.
Bacteria often attach to surfaces and form dense aggregations called biofilms or bacterial mats. These films can range from a few micrometers in thickness to up to half a meter in depth, and may contain multiple species of bacteria, protists and archaea. Bacteria living in biofilms display a complex arrangement of cells and extracellular components, forming secondary structures such as microcolonies, through which there are networks of channels to enable better diffusion of nutrients. In natural environments, such as soil or the surfaces of plants, the majority of bacteria are bound to surfaces in biofilms. Biofilms are also important for chronic bacterial infections and infections of implanted medical devices, as bacteria protected within these structures are much harder to kill than individual bacteria. pharma .blogspot.com
Even more complex morphological changes are sometimes possible. For example, when starved of amino acids, Myxobacteria detect surrounding cells in a process known as quorum sensing, migrate towards each other, and aggregate to form fruiting bodies up to 500 micrometres long and containing approximately 100,000 bacterial cells. In these fruiting bodies, the bacteria perform separate tasks; this type of cooperation is a simple type of multicellular organisation. For example, about one in 10 cells migrate to the top of these fruiting bodies and differentiate into a specialised dormant state called myxospores, which are more resistant to desiccation and other adverse environmental conditions than are ordinary cells.

Intracellular structures

The bacterial cell is surrounded by a lipid membrane, or cell membrane, which encompasses the contents of the cell and acts as a barrier to hold nutrients, proteins and other essential components of the cytoplasm within the cell. As they are prokaryotes, bacteria do not tend to have membrane-bound organelles in their cytoplasm and thus contain few intracellular structures. They consequently lack a nucleus, mitochondria, chloroplasts and the other organelles present in eukaryotic cells, such as the Golgi apparatus and endoplasmic reticulum. However, recent research is identifying increasing amounts of structural complexity in bacteria, such as the discovery of the prokaryotic cytoskeleton.
Many important biochemical reactions, such as energy generation, occur due to concentration gradients across membranes, creating a potential difference analogous to a battery. The absence of internal membranes in bacteria means these reactions, such as electron transport, occur across the cell membrane, between the cytoplasm and the periplasmic space. Additionally, while some transporter proteins consume chemical energy, others harness concentration gradients to import nutrients across the cell membrane or to expel undesired molecules from the cytoplasm.
Bacteria do not have a membrane-bound nucleus, and their genetic material is typically a single circular chromosome located in the cytoplasm in an irregularly shaped body called the nucleoid. The nucleoid contains the chromosome with associated proteins and RNA. Like all living organisms, bacteria contain ribosomes for the production of proteins, but the structure of the bacterial ribosome is different from those of eukaryotes and Archaea.The order Planctomycetes are an exception to the general absence of internal membranes in bacteria, because they have a membrane around their nucleoid and contain other membrane-bound cellular structures.
Some bacteria produce intracellular nutrient storage granules, such as glycogen,polyphosphate, sulfur or polyhydroxyalkanoates. These granules enable bacteria to store compounds for later use. Certain bacterial species, such as the photosynthetic Cyanobacteria, produce internal gas vesicles, which they use to regulate their buoyancy - allowing them to move up or down into water layers with different light intensities and nutrient levels.
Extracellular structuresAround the outside of the cell membrane is the bacterial cell wall. Bacterial cell walls are made of peptidoglycan (called murein in older sources), which is made from polysaccharide chains cross-linked by unusual peptides containing D-amino acids. Bacterial cell walls are different from the cell walls of plants and fungi, which are made of cellulose and chitin, respectively. The cell wall of bacteria is also distinct from that of Archaea, which do not contain peptidoglycan. The cell wall is essential to the survival of many bacteria, and the antibiotic penicillin is able to kill bacteria by inhibiting a step in the synthesis of peptidoglycan.
There are broadly speaking two different types of cell wall in bacteria, called Gram-positive and Gram-negative. The names originate from the reaction of cells to the Gram stain, a test long-employed for the classification of bacterial species.
Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and lipoproteins. Most bacteria have the Gram-negative cell wall, and only the Firmicutes and Actinobacteria (previously known as the low G+C and high G+C Gram-positive bacteria, respectively) have the alternative Gram-positive arrangement.These differences in structure can produce differences in antibiotic susceptibility; for instance, vancomycin can kill only Gram-positive bacteria and is ineffective against Gram-negative pathogens, such as Haemophilus influenzae or Pseudomonas aeruginosa.In many bacteria an S-layer of rigidly arrayed protein molecules covers the outside of the cell.This layer provides chemical and physical protection for the cell surface and can act as a macromolecular diffusion barrier. S-layers have diverse but mostly poorly understood functions, but are known to act as virulence factors in Campylobacter and contain surface enzymes in Bacillus stearothermophilus.
Flagella are rigid protein structures, about 20 nanometres in diameter and up to 20 micrometres in length, that are used for motility. Flagella are driven by the energy released by the transfer of ions down an electrochemical gradient across the cell membrane.Fimbriae are fine filaments of protein, just 2–10 nanometres in diameter and up to several micrometers in length. They are distributed over the surface of the cell, and resemble fine hairs when seen under the electron microscope. Fimbriae are believed to be involved in attachment to solid surfaces or to other cells and are essential for the virulence of some bacterial pathogenPili (sing. pilus) are cellular appendages, slightly larger than fimbriae, that can transfer genetic material between bacterial cells in a process called conjugation (see bacterial genetics, below).
Capsules or slime layers are produced by many bacteria to surround their cells, and vary in structural complexity: ranging from a disorganised slime layer of extra-cellular polymer, to a highly structured capsule or glycocalyx. These structures can protect cells from engulfment by eukaryotic cells, such as macrophages. They can also act as antigens and be involved in cell recognition, as well as aiding attachment to surfaces and the formation of biofilms.
The assembly of these extracellular structures is dependent on bacterial secretion systems. These transfer proteins from the cytoplasm into the periplasm or into the environment around the cell. Many types of secretion systems are known and these structures are often essential for the virulence of pathogens, so are intensively studied.
Endospores
Certain genera of Gram-positive bacteria, such as Bacillus, Clostridium, Sporohalobacter, Anaerobacter and Heliobacterium, can form highly resistant, dormant structures called endospores.In almost all cases, one endospore is formed and this is not a reproductive process, although Anaerobacter can make up to seven endospores in a single cell.Endospores have a central core of cytoplasm containing DNA and ribosomes surrounded by a cortex layer and protected by an impermeable and rigid coat.
Endospores show no detectable metabolism and can survive extreme physical and chemical stresses, such as high levels of UV light, gamma radiation, detergents, disinfectants, heat, pressure and desiccation.In this dormant state, these organisms may remain viable for millions of years,and endospores even allow bacteria to survive exposure to the vacuum and radiation in space.Endospore-forming bacteria can also cause disease: for example, anthrax can be contracted by the inhalation of Bacillus anthracis endospores, and contamination of deep puncture wounds with Clostridium tetani endospores causes tetanus.

WHAT IS BACTERIA

Bacteria (singular: bacterium) are unicellular microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods to spirals. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, radioactive waste, seawater, and deep in the Earth's crust. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water; in all, there are approximately five nonillion (5×1030) bacteria on Earth, forming much of the world's biomass. Bacteria are vital in recycling nutrients, and many important steps in nutrient cycles depend on bacteria, such as the fixation of nitrogen from the atmosphere. However, most of these bacteria have not been characterized, and only about half of the phyla of bacteria have species that can be cultured in the laboratory. The study of bacteria is known as bacteriology, a branch of microbiology.
There are approximately ten times as many bacterial cells as human cells in the human body, with large numbers of bacteria on the skin and in the digestive tract. Although the vast majority of these bacteria are rendered harmless or beneficial by the protective effects of the immune system, a few are pathogenic bacteria and cause infectious diseases, including cholera, syphilis, anthrax, leprosy and bubonic plague. The most common fatal bacterial diseases are respiratory infections, with tuberculosis alone killing about 2 million people a year, mostly in sub-Saharan Africa. In developed countries, antibiotics are used to treat bacterial infections and in various agricultural processes, so antibiotic resistance is becoming common. In industry, bacteria are important in processes such as sewage treatment, the production of cheese and yoghurt, and the manufacture of antibiotics and other chemicals. pharmaguideline.
Bacteria are prokaryotes. Unlike cells of animals and other eukaryotes, bacterial cells do not contain a nucleus and rarely harbour membrane-bound organelles. Although the term bacteria traditionally included all prokaryotes, the scientific classification changed after the discovery in the 1990s that prokaryotic life consists of two very different groups of organisms that evolved independently from an ancient common ancestor. These evolutionary domains are called Bacteria and Archaea.
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History of Microbiology

Discovery and origins of microbiologyBacteria and microorganisms were first observed by Antonie van Leeuwenhoek in 1676 using a single-lens microscope of his own design. In doing so Leeuwenhoek made one of the most important discoveries in biology and initiated the scientific fields of bacteriology and microbiology.[1] The name "bacterium" was introduced much later, by Ehrenberg in 1828, derived from the Greek βακτηριον meaning "small stick". While Van Leeuwenhoek is often cited as the first microbiologist, the first recorded microbiological observation, that of the fruiting bodies of molds, was made earlier in 1665 by Robert Hooke.
The field of bacteriology (later a subdiscipline of microbiology) is generally considered to have been founded by Ferdinand Cohn (1828–1898), a botanist whose studies on algae and photosynthetic bacteria led him to describe several bacteria including Bacillus and Beggiatoa. Cohn was also the first to formulate a scheme for the taxonomic classification of bacteria. Louis Pasteur (1822–1895) and Robert Koch (1843–1910) were contemporaries of Cohn’s and are often considered to be the founders of medical microbiology. Pasteur is most famous for his series of experiments designed to disprove the then widely held theory of spontaneous generation, thereby solidifying microbiology’s identity as a biological science.[10] Pasteur also designed methods for food preservation (pasteurization) and vaccines against several diseases such as anthrax, fowl cholera and rabies. Koch is best known for his contributions to the germ theory of disease, proving that specific diseases were caused by specific pathogenic microorganisms. He developed a series of criteria that have become known as the Koch's postulates. Koch was one of the first scientists to focus on the isolation of bacteria in pure culture resulting in his description of several novel bacteria including Mycobacterium tuberculosis, the causative agent of tuberculosis.
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While Pasteur and Koch are often considered the founders of microbiology, their work did not accurately reflect the true diversity of the microbial world because of their exclusive focus on microorganisms having direct medical relevance. It was not until the work of Martinus Beijerinck (1851–1931) and Sergei Winogradsky (1856–1953), the founders of general microbiology (an older term encompassing aspects of microbial physiology, diversity and ecology), that the true breadth of microbiology was revealed. Beijerinck made two major contributions to microbiology: the discovery of viruses and the development of enrichment culture techniques.[11] While his work on the Tobacco Mosaic Virus established the basic principles of virology, it was his development of enrichment culturing that had the most immediate impact on
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microbiology by allowing for the cultivation of a wide range of microbes with wildly different physiologies. Winogradsky was the first to develop the concept of chemolithotrophy and to thereby reveal the essential role played by microorganisms in geochemical processes. He was responsible for the first isolation and description of both nitrifying and nitrogen-fixing bacteria.

Microbiology Introduction

Microbiology is the study of microorganisms, which are unicellular or cell-cluster microscopic organisms. This includes eukaryotes such as fungi and protists, and prokaryotes such as bacteria and certain algae. Viruses, though not strictly classed as living organisms, are also studied. Microbiology is a broad term which includes many branches like virology, mycology, parasitology and others. A person who specializes in the area of microbiology is a microbiologist.
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Although much is now known in the field of microbiology, advances are being made regularly. We have probably only studied about one percent of all of the microbe species on Earth. Thus, despite the fact that over three hundred years have passed since the discovery of microbes, the field of microbiology could be said to be in its infancy relative to other biological disciplines such as zoology, botany and entomology.