Glassware sterilization

Normal laboratory glassware must first be washed and cleaned. It has to be rinsed with deionised water. The clean glassware is sterilised in an oven set at 200 °C for 1 1 hours. It is suitable to cover glassware with aluminum foil to maintain aseptic conditions after removing the glassware from the oven. If aluminum foil is not available, special heat-resistant wrap paper can be used. The sterile glassware must be protected from the air, which has micro-flora, or any contaminants. Avoid the use of any plastic caps and papers. Detach any labelling tape or other flammable materials, as they are fire hazards. 

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. 

Dry heat is widely used to sterilize glassware and equipment parts in manufacturing areas for parenteral products. It has good penetration power and is not as corrosive as steam. However, heat-up time is... 

Sterilizer Sterilization of culture plates and glasswares Trained Free... 

Subsequent to DNA template linearization, the procedures for in vitro RNA transcription reaction are set up. It is essential to avoid RNase contamination by using gloves, sterile glassware, and water devoid of RNase activity [treated with 0.1% diethyl pyrocarbonate (DEPC) and autoclaved]. 

The equipment needed is determined by the type and extent of the services chosen to provide. Hospitals already utilize laminar flow hoods for aseptic compounding of sterile solutions. The same hoods can be used to compound other sterile products such as eye drops. A balance, preferably electronic, is essential. Ointment slabs (pill tiles), along with spatulas of different types and materials, should be on hand. A few mortars and pestles (both of glass, ceramic, and/or plastic) should be obtained and some glassware. It may not be necessary to buy a roomful of equipment, but one should purchase what is needed to start the service, and build it up as the service grows and expands to different arenas. 

The water used in all solutions and to wash and rinse the glassware should be of the highest purity possible. Sterile deionized glass-distilled water is recommended for all procedures. 

Fill washed and dried glassware with sterile filtered water, sonicate for 5 minutes and test with a particle counter. 

Samples should be collected aseptically in nonpyrogenic containers. Reused depyrogenate glassware or sterile, disposable polystyrene plastics are recommended to minimize adsorption of endotoxin. 

Heat is the most widely used means of sterilization, which can be employed for both liquid medium and heatable solid objects. It can be applied as dry or moist heat (steam). The moist heat is more effective than the dry heat, because the intrinsic heat resistance of vegetative bacterial cells is greatly increased in a completely dry state. As a result the death rate is much lower for the dry cells than for moist ones. The heat conduction in dry air is also less rapid than in steam. Therefore, dry heat is used only for the sterilization of glassware or heatable solid materials. By pressurizing a vessel, the steam temperature can be increased significantly above the boiling point of water. Laboratory autoclaves are commonly operated at a steam pressure of about 30 psia, which corresponds to 121°C. Even bacterial spores are rapidly killed at 121 °C. 

Washing and sterilizing glassware using proper procedures. 

The methods of preparation of glassware are indicated in Chapter 8, and if sterilisation is monitored as described the glassware should not be a source of contamination. Likewise plasticware is obtained from the manufacturer in a sterile condition. Usually sterilisation of plastic is achieved using ethylene oxide or irradiation procedures and vessels are supplied wrapped in cellophane. 

Note Ensure that all glassware and supplies are heat sterilized. The work area should be aseptically clean. 

The following procedures require sterile technique that is, use of sterilized glassware, pipettes, Petri plates, and other equipment. Do not use equipment that has lost a covering cap and consequently has been exposed to the air for long periods. If you are unfamiliar with sterile techniques for transferring bacterial cultures, consult your instructor for details of the techniques required in this experiment. 

The enzyme, a protein, is highly susceptible to attack by airborne microorganisms. All glassware that comes in contact with the enzyme must be cleaned and rinsed very carefully. Place about 50 mL of the enzyme solution in a glass-stoppered Erlenmeyer flask, and keep this flask chilled with ice throughout the experiment. Any dilution of the enzyme stock solution must be done with chilled water that has been previously sterilized by boiling. 

Dry heat sterilization is used to depyrogenate glassware and other heat-stable mateiials. Temperatures in the range of 250°-325°C rapidly inactivate endotoxin by thermal incineration. The size and mass of a load influence the time required to reach equilibrium within the oven therefore, empirical data are required to verify that desired conditions were achieved. Overkill cycles in the range of 4-6 log reduction values (LRV) are common. A heat exposure of 250°C for at least 30 min provides greater than a 3 log reduction. 

Molin, G. Molin, O. Dry-heat sterilization of pharmaceutical glassware using hot air or infra-red radiation. Acta Pharm. Suec. 1976,13, 476 481-483. 

Dry heat application is generally restricted to glassware and metal surgical instruments (where its good penetrability and non-corrosive nature are of benefit), non-aqueous thermostable liquids and thermostable powders (see Chapter 19). In practice, the range of materials that are actually subjected to dry heat sterilization is quite limited, and... 

Ensure that all glassware and plasticware are DNase- and RNase-free grade. The use of DNase- and RNase-free sterile, disposable, plastic tubes is recommended throughout the procedure. 

It is a sine qua non to avoid any contamination with RNases. Sterile, disposable plasticware is essentially RNase-free and should be used whenever possible. General laboratory glassware, however, should be treated by baking at 180°C overnight, or, in the case of... 

Under this heading one often finds basic issues such as selection and maintenance of equipment and reagents, washing of glassware, making of buffers and other solutions, sterilization, use of pipettes, etc. These are perhaps the most obvious prescriptions. They are fundamental to any study in biochemistry, and thus one finds them emphasized in beginning laboratory courses. 

Great care should be taken to ensure that all apparatus and chemicals are sterile and free from RNase contamination. Glassware should be incubated at above 150°C for at least 6 h. All solutions not containing amines should be treated with diethyl-pyrocarbonate (DEPC) and autoclaved. If solutions contain amines (Tris), RNase-free chemicals should be added to DEPC-treated distilled water. Gloves should be worn at all times and changed frequently. 

Chemical and glassware storage, dish washing, sample refrigerators, glassware dryers, autoclaves for the preparation of sterile sample bottles for the plant, computer(s) for assay calculations, water baths, fume hoods, etc., are additional basic equipment items needed. Typical overall space requirements are 450 ft of floor space per working chemical technician. 

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