04_virology_cell_culture_laboratory
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures Applied Veterinary Virology: The isolation and identification of viruses using cell cultures Authors: Prof Estelle Venter Licensed under a Creative Commons Attribution license. CELL CULTURE LABORATORY The major item required by the cell culture scientist is a laminar flow cabinet or a sterile working hood. However, such cabinets should not be seen as an insurance policy against poor technique. A cabinet should be selected so that it has sufficient working space for manipulating the cell system being used. Remember that there must be room for media, sera, additive bottles, pipettes and measuring devices, provision for gassing cultures where necessary, room for storage of the culture vessels, plus an appropriate place for placing discards. Basic requirements Incubator: If bicarbonate buffering systems are to be used, an incubator containing CO2 is essential Inverted microscope Water baths: one at 37°C and the other at 56°C (for inactivating sera) Centrifuge – normally low speed Pipetting devices Filtration equipment - For filtering media Counting apparatus - Haemocytometer or Coulter counter, if cell counting is to be done Storage of cells - Tubes for storage, as well as an appropriate deep freezer or liquid nitrogen container Safety cabinets - Safety cabinets are either use to protect the substance which one is working with or the operator or both. Potential infectious material should always be handled where possible in a biohazard cabinet. Laminar flow cabinets: Creates a sterile environment for aseptic work Prevents contamination of culture media during pouring and tubing It should not be used for the following: Manipulation of pathogenic or hazardous substances 1|Page Applied Veterinary Virology: The isolation and identification of viruses using cell cultures Dispensing of tissue culture cells or any other living material; because even sterile culture cells may contain undetectable slow or oncogenic viruses, which are blown towards the operator’s face or into the room Biological safety cabinets: Mainly for protection of the operator working with, e.g. Pathogenic organism Highly concentrated biological agents known to be hazardous to humans, animals or plants Mutagens Low and moderate risk oncogenic viruses Functions: These cabinets are divided into different classes according to its way of functioning i.e. CLASS I, CLASS II and CLASS III biosafety cabinets. Class I biosafety cabinet A class 1 cabinet is an open-fronted, negative pressure (drawing air away from the operator), with a HEPA-filtered re-circulated air exhaust, which is suitable for class B agents (bacteria). The operator should always wear gloves. This cabinet is mainly for the protection of the operator against air borne infections. Class II biosafety cabinet A class II cabinet is a vertical-laminar flow cabinet with HEPA-filtered re-circulated airflow in the work area and HEPA-filtered air exhaust. These cabinets are useful for the containment of BSL 2 and 3 agents. This is of particular value in virological procedures, such as work with tissue cultures and embryonated eggs because it offers protection for both the operator and the work against contamination. Class III biosafety cabinet These are used mainly in level 4 or maximum containment Labs where very harzadous viruses are investigated. Class III cabinets are the ultimate in safety cabinet engineering; they are completely enclosed and air tight. The operator works through gloves fitted in the see through front window. Cleaning of tissue culture items General procedure for items that do not come into direct contact with cells All items should be soaked in disinfectant immediately after use Wash properly Rinse in tap water Rinse separately in fresh distilled water 2|Page Applied Veterinary Virology: The isolation and identification of viruses using cell cultures Air dry thoroughly Cover and assemble for sterilization; date of sterilization should be clearly marked General procedure for items that come into direct contact with cells Disposable plastic ware should be thrown into separate bags for autoclaving and decontamination Reusable glassware must be decontaminated in a disinfectant, e.g. chlorine solution, and then autoclaved Plastic ware Tissue culture flasks, plastic disposable pipettes, syringes, etc. Culture surface must be totally non-toxic and uniform. Pre-tested batches of disposable tissue culture flasks are generally used. Table 1: Disposal of laboratory material Material Disposal route Specimen containers Plastic disposable Incinerator bucket incinerate Glass autoclave drumautoclave discard Laboratory glassware Bottles, tubes and vials Firmly closed autoclave drumautoclave recycle or discard Open tubes Upright in basket autoclave drumautoclave recycle Pyrex centrifugation tubes Immerse overnight in corox autoclave drumautoclave Electron microscope glassware recycle Broken glassware Plastic bag sealadditional wrapping and separate autoclavediscard Laboratory plastics Disposable: cellulose nitrate tubes, plastic Plastic bag seal incinerate haemagglutination and microtiter plates Recycle Soak in decon or coroxrinserecycle (radiation) Pipettes Glass Immerse in corox / decon washautoclave Plastic Plastic bag seal incinerate Rubber stoppers Cell culture stoppers Overnight in corox washcover with foil or autoclave plastic bagautoclave recycle Instruments 3|Page Applied Veterinary Virology: The isolation and identification of viruses using cell cultures Scissors, forceps, clamps, mortars and pestles, grinders, Immerse in water with corox or decon wash autoclave homogenizers recycle Uninfected tissue Placentas, kidneys, embryonic tissues Plastic bagsealincinerate Contaminated tissue PM specimens, inoculated egg material Seal in containerbagautoclavediscard Protective clothing Gowns Bagsclose securelylaunder Plastic aprons, gloves, etc. Plastic bag (sealed)incinerate Paper products Paper towels, tissue, etc. 4|Page Plastic bag (tie securely) incinerate
