A medical laboratory or clinical laboratory is a laboratory where tests are done on biological specimens in order to get information about the health of a patient.
The laboratory is often divided into a number of disciplines:
Microbiology receives swabs, feces, urine, blood, sputum, medical equipment, as well as possible infected tissue. They culture this to check for any pathogenic microbes.
Parasitology investigates parasites.
Hematology receives whole blood and citrated plasma. They do full blood counts, and blood films.
Coagulation analyzes clotting times and coagulation factors.
Clinical chemistry usually receives serum. They test the serum for different components.
Toxicology tests for pharmaceutical drugs, drugs of abuse or recreational use, and other toxins.
Immunology tests for antibodies.
Serology receives serum samples to look for evidence of diseases such as hepatitis or HIV.
Urinalysis tests urine for many analytes
Histology processes solid tissue removed from the body to make slides and examine cellular detail.
Cytology examines smears of cells (such as from the cervix) for evidence of cancer and other conditions.
Cytogenetics involves using blood and other cells to get a karyotype. This can be helpful in prenatal diagnosis (e.g. Down's syndrome) as well as in cancer (some cancers have abnormal chromosomes).
Virology and DNA analysis are also done in large medical laboratories.
Surgical pathology examines organs, limbs, tumors, fetuses, and other tissues biopsied in surgery such as breast masectomys.
Thursday, October 11, 2007
Sunday, September 09, 2007
Autoclave
Autoclave Action
Sterilization is defined as the complete destruction of all forms of microbial life, including bacterial spores. The meaning of this word is absolute; there is no such thing as "partial sterilization." Something is either sterile or non-sterile. Sterilization can be accomplished by either physical or chemical means. The principal physical means is autoclaving; other physical methods include boiling and dry heat. Chemicals used for sterilization include the gases ethylene oxide and formaldehyde, and liquids such as glutaraldehyde. Of all these sterilants, autoclaving is the fastest and most reliable.
Why is an autoclave such an effective sterilizer? An autoclave is a large pressure cooker; it operates by using steam under pressure as the sterilizing agent. High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100º C has almost seven times more heat than boiling water. Steam is able to penetrate objects with cooler temperatures because once the steam contacts a cooler surface it immediately condenses to water, producing a concomitant 1,870 fold decrease in steam volume. This creates negative pressure at the point of condensation and draws more steam to the area. Condensations continues so long as the temperature of the condensing surface is less than that of steam. These properties ensure rapid heating of surfaces, good penetration of dense materials, and coagulation of proteins.
Moist heat is thought to kill microorganisms by causing coagulation of essential proteins. Death rate is directly proportional to the concentration of microorganisms at any given time. The time required to kill a known population of microorganisms in a specific suspension at a particular temperature is referred to as thermal death time (TDT). Increasing the temperature decreases TDT, and lowering the temperature increases TDT. Processes conducted at high temperatures for short periods of time are preferred over lower temperatures for longer times.
Environmental conditions also influence TDT. Increased heat causes increased toxicity of metabolic products and toxins. TDT decreases with pronounced acidic or basic pHs. However, fats and oils slow heat penetration and increase TDT. It must be remembered that thermal death times are not precise values; they measure the effectiveness and rapidity of a sterilization process.
Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/temperature relationship. These two variables are extremely important. Higher temperatures ensure more rapid killing. Some standard temperature/pressures employed are 115ºC/10 p.s.i., 121ºC/15 p.s.i., and 132ºC/27 p.s.i. Longer times are needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic media, liquids, and many other things. However, certain items, such as fiber-optic endoscopes, cannot withstand autoclaving and should be sterilized with chemical or gas sterilants. When proper conditions and time are employed, no living organisms will survive a trip through an autoclave.
Sterilization is defined as the complete destruction of all forms of microbial life, including bacterial spores. The meaning of this word is absolute; there is no such thing as "partial sterilization." Something is either sterile or non-sterile. Sterilization can be accomplished by either physical or chemical means. The principal physical means is autoclaving; other physical methods include boiling and dry heat. Chemicals used for sterilization include the gases ethylene oxide and formaldehyde, and liquids such as glutaraldehyde. Of all these sterilants, autoclaving is the fastest and most reliable.
Why is an autoclave such an effective sterilizer? An autoclave is a large pressure cooker; it operates by using steam under pressure as the sterilizing agent. High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100º C has almost seven times more heat than boiling water. Steam is able to penetrate objects with cooler temperatures because once the steam contacts a cooler surface it immediately condenses to water, producing a concomitant 1,870 fold decrease in steam volume. This creates negative pressure at the point of condensation and draws more steam to the area. Condensations continues so long as the temperature of the condensing surface is less than that of steam. These properties ensure rapid heating of surfaces, good penetration of dense materials, and coagulation of proteins.
Moist heat is thought to kill microorganisms by causing coagulation of essential proteins. Death rate is directly proportional to the concentration of microorganisms at any given time. The time required to kill a known population of microorganisms in a specific suspension at a particular temperature is referred to as thermal death time (TDT). Increasing the temperature decreases TDT, and lowering the temperature increases TDT. Processes conducted at high temperatures for short periods of time are preferred over lower temperatures for longer times.
Environmental conditions also influence TDT. Increased heat causes increased toxicity of metabolic products and toxins. TDT decreases with pronounced acidic or basic pHs. However, fats and oils slow heat penetration and increase TDT. It must be remembered that thermal death times are not precise values; they measure the effectiveness and rapidity of a sterilization process.
Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/temperature relationship. These two variables are extremely important. Higher temperatures ensure more rapid killing. Some standard temperature/pressures employed are 115ºC/10 p.s.i., 121ºC/15 p.s.i., and 132ºC/27 p.s.i. Longer times are needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic media, liquids, and many other things. However, certain items, such as fiber-optic endoscopes, cannot withstand autoclaving and should be sterilized with chemical or gas sterilants. When proper conditions and time are employed, no living organisms will survive a trip through an autoclave.
Thursday, September 06, 2007
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 - 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 1950's, 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.[3]
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.[4] 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 lenghthen 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 weeks), and is expensive. Formaldehyde is less expensive and has a much longer shelf life if some methanol is added to inhibit polymerization to paraformaldehyde, but is much more volatile. Formaldehyde is also used as a gaseous sterilizing agent; in this case, it is prepared on-site by depolymerization of solid paraformaldehyde. Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde.
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.
Sterrad 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. 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.
Radiation sterilization
Methods exist to sterilize using radiation such as X-rays, gamma rays, or subatomic particles.
Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, canulas 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).
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).
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.
One may also use a "flowbox", a device which produces a laminar stream of air flowing downwards, kept at constant temperature by special air conditioning. It is particullary important when working with pure cultures.
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 - 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 1950's, 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.[3]
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.[4] 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 lenghthen 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 weeks), and is expensive. Formaldehyde is less expensive and has a much longer shelf life if some methanol is added to inhibit polymerization to paraformaldehyde, but is much more volatile. Formaldehyde is also used as a gaseous sterilizing agent; in this case, it is prepared on-site by depolymerization of solid paraformaldehyde. Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde.
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.
Sterrad 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. 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.
Radiation sterilization
Methods exist to sterilize using radiation such as X-rays, gamma rays, or subatomic particles.
Gamma rays are very penetrating and are commonly used for sterilization of disposable medical equipment, such as syringes, needles, canulas 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).
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).
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.
One may also use a "flowbox", a device which produces a laminar stream of air flowing downwards, kept at constant temperature by special air conditioning. It is particullary important when working with pure cultures.
Heat sterilization
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 innoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area.
Incineration will also burn any 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 15 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 anyone most anywhere, requiring only water, enough heat, and a container that can withstand the heat; however, boiling can be hazardous and cumbersome.
Tindalization[1] /Tyndallization[2] 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.
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 treatement. However, sodium hydroxide may corrode surgical instruments, especially at the elevated temperatures of the autoclave.
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 innoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area.
Incineration will also burn any 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 15 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 anyone most anywhere, requiring only water, enough heat, and a container that can withstand the heat; however, boiling can be hazardous and cumbersome.
Tindalization[1] /Tyndallization[2] 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.
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 treatement. However, sodium hydroxide may corrode surgical instruments, especially at the elevated temperatures of the autoclave.
Sterilization
Sterilization (or sterilisation) 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, or filtration.
Medicine and surgery
In 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.
Heat sterilization
Steam sterilization
A widely-used method for heat sterilization is the autoclave. Autoclaves commonly use steam heated to 121°C (250°F), at 103 kPa (15 psi) above atmospheric pressure. Solid surfaces are effectively sterilized when heated this temperature for at least 15 minutes or to 134°C for a minimum of 3 minutes. However, liquids and instruments packed in layers of cloth require a much longer time to reach a sterilizing temperature. After sterilization, autoclaved liquids must be cooled slowly to avoid boiling over when the pressure is released.
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.
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 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 allowed to escape as steam enters the autoclave chamber; otherwise the final temperature will be less than that of the entering steam. 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.
Medicine and surgery
In 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.
Heat sterilization
Steam sterilization
A widely-used method for heat sterilization is the autoclave. Autoclaves commonly use steam heated to 121°C (250°F), at 103 kPa (15 psi) above atmospheric pressure. Solid surfaces are effectively sterilized when heated this temperature for at least 15 minutes or to 134°C for a minimum of 3 minutes. However, liquids and instruments packed in layers of cloth require a much longer time to reach a sterilizing temperature. After sterilization, autoclaved liquids must be cooled slowly to avoid boiling over when the pressure is released.
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.
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 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 allowed to escape as steam enters the autoclave chamber; otherwise the final temperature will be less than that of the entering steam. 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.
An electrode is an electrical conductor used to make contact with a nonmetallic part of a circuit (e.g. a semiconductor, an electrolyte or a vacuum). The word was coined by the scientist Michael Faraday from the Greek words elektron (meaning amber, from which the word electricity is derived) and hodos, a way.
Polarizable and Non-Polarizable Electrodes :
Perfectly Polarizable Electrodes :
These are electrodes in which no actual charge crosses the electrode-electrolyte interface when a current is applied. The current across the interface is a displacement current and the electrode behaves like a capacitor. Example : Ag/AgCl Electrode
Perfectly Non-Polarizable Electrode
These are electrodes where current passes freely across the electrode-electrolyte interface, requiring no energy to make the transition. These electrodes see no overpotentials. Example : Platinum electrode
Commonly Used Biopotential Electrodes :
Metal plate electrodes :
–Large surface: Ancient, therefore still used, ECG
–Metal disk with stainless steel; platinum or gold coated
–EMG, EEG
–smaller diameters
–motion artifacts
–Disposable foam-pad: Cheap
Suction Cup electrodes
- No straps or adhesives required
- precordial (chest) ECG
- can only be used for short periods
Floating electrodes
- metal disk is recessed
- swimming in the electrolyte gel
- not in contact with the skin
- reduces motion artifact
Needle and wire electrodes for percutaneous measurement of biopotentials
Fetal ECG Electrodes :
Electrodes for detecting fetal electrocardiogram during labor, by means of intracutaneous needles (a) Suction electrode. (b) Cross-sectional view of suction electrode in place, showing penetration of probe through epidermis. (c) Helical electrode, which is attached to fetal skin by corkscrew type action.
Wednesday, September 05, 2007
Electrodes
The human body's nervous or muscular system uses the ebb and flow of ions to communicate. This ionic transport within and along the nerve fibers can be measured on the surface of the skin using a specific type of electrochemical sensor commonly referred to as the surface recording electrode (sometimes just called the electrode).
The purpose of the electrode is to act as a transducer between the ionic transport of the nerve/ muscle and the electron flow in copper wire. It is the junction between the electrode and the electrolyte that allows such a transduction to take place.
Monday, August 20, 2007
pH METER PRINCIPLE OF WORKING
Introduction to pH
pH is a unit of measure which describes the degree of acidity or alkalinity of a solution. It is measured on a scale of 0 to 14. The term pH is derived from "p", the mathematical symbol of the negative logarithm, and "H", the chemical symbol of Hydrogen. The formal definition of pH is the negative logarithm of the Hydrogen ion activity.
pH Measurement A rough indication of pH can be obtained using pH papers or indicators, which change color as the pH level varies. These indicators have limitations on their accuracy, and can be difficult to interpret correctly in colored or murky samples.
More accurate pH measurements are obtained with a pH meter. A pH measurement system consists of three parts: a pH measuring electrode, a reference electrode, and a high input impedance meter. The pH electrode can be thought of as a battery, with a voltage that varies with the pH of the measured solution. The pH measuring electrode is a hydrogen ion sensitive glass bulb, with a millivolt output that varies with the changes in the relative hydrogen ion concentration inside and outside of the bulb. The reference electrode output does not vary with the activity of the hydrogen ion. The pH electrode has very high internal resistance, making the voltage change with pH difficult to measure. The input impedance of the pH meter and leakage resistances are therefore important factors. The pH meter is basically a high impedance amplifier that accurately measures the minute electrode voltages and displays the results directly in pH units on either an analog or digital display. In some cases, voltages can also be read for special applications or for use with ion-selective or Oxidation-Reduction Potential (ORP) electrodes.
PLEASE CLICK ON THE FOLLOWING SITE FOR AN ANIMATION ON THE pH METER
Thursday, August 02, 2007
Colorimeter (spectrophotometer)
Many tests are done on blood to detect diseases e.g. diabetes, hypercholesterolemia,etc.
A nice animation is shown on the site below:
http://virtuallaboratory.net/Focussed%20Labs/Dilution/section_05.html
A nice animation is shown on the site below:
http://virtuallaboratory.net/Focussed%20Labs/Dilution/section_05.html
Tuesday, July 31, 2007
Working of the Blood Cell Counter
Click on the site to see a nice animation of the Flow Cytometer. It is one of the automated ways to measure blood cells:
http://www.unsolvedmysteries.oregonstate.edu/flow_cytometry_06.shtml
You cane also read Page 351 of the textbook (Cromwell, Weibell and Pfeiffer) and refer to Fig. 13.4
http://www.unsolvedmysteries.oregonstate.edu/flow_cytometry_06.shtml
You cane also read Page 351 of the textbook (Cromwell, Weibell and Pfeiffer) and refer to Fig. 13.4
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