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Kuliah 7, Equipment and Biosafety in Cell Culture laboratory

Kuliah 7, Equipment and Biosafety in Cell Culture laboratory

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06/28/2012

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Equipment and Biosafety in Cell Culture Laboratory

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Requirement - Essential - Beneficial - Useful

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Laminar flow hood Usually, one hood is suffient for two or three people. Horizontal flow hood Cheaper and provide the best sterile protection for cell culture

Suitable only for preparing medium. . other sterile reagents and culturing nonprimate cells Potentially hazardous materials. a Class II or Class III biohazard cabinet should be used Important to familiarize yourself with local and national biohazard regulations before installing equipment.

Quite (noisy hoods are more fatiguing) .‡ Choose a Hood .Comfortable to sit . .Large enough . lowered.Front screen should be able to be raised. or removed completely.Easily clean both inside the working area and below the work surface in the event of spillage .

Many incubator have a heated water jacket to distribute heat evenly around cabinet.` CO2 Incubator Double chamber is preferable to one large incubator it can accommodate more cultures with better temperature control. thus avoiding the formation of cold spot .

` More expensive. and injects pure CO2 into the incubator to make up any deficiency. but their ease of use and superiors control of temperature and CO2 tension. Determine the concentration of CO2. ` .

Leave sufficient space around the sterilizer for maintenance and ventilation .` Sterilizer The simplest and cheapest sterilizer is a domestic pressure cooker that generates 100 kpa above ambient. Bencth top autoclave give automatic programming and safety locking.

Cold Room is easier to access more economical in terms of space than are several separate refrigerators .` Refrigerator and Freezers Domestic refrigerator or freezer is quite efficient and cheaper than special laboratory equipment.

` Inverted microscope Large enough to accommodate culture dish Invest in microscopes with a high quality optics. . with provisions for a camera. long working distance phase contrast condense and objective.

and diluting concentrates. Deionized or reverse water. ` . Water Purifications Required for rinsing glassware.` Washing Up Soaking baths or sinks should be deep enough so that all your glassware can be totally immersed in detergent during soaking. ultrapure water. dissolving powdered media.

` Centrifuge Periodiccaly. Small bench top centrifuge. . preferably with proporsionally controlled braking is suffient for most purposes. cell suspensions require centrifugations to increase the concentration of cells or to wash off reagent.

Economy and static holding time c. Convinience of access of contents Liquid Nitrogen Tank - .The choice of freezer is determined by three factors: a.` Cryostorage Container . Capacity b.

` Balance Hemocytometer It is essential to have some means of counting cells. Hemocytometer slide is the cheapest option and has the added benefit of allowing cell viability to be determined by dye exclusion ` .

` ` ` ` ` ` ` Cell counter Aspiration pump pH meter Conductivity Meter Osmometer Upright microscope Dissecting microscope .

` ` ` ` Temperature Recording Magnetic Stirrer Roller Racks Fluid handling Removal of fluid Nonsterile dispensing Simple pippeting Pipettors .

` ` ` ` ` ` ` Low temperature Freezer Glassware washing Machine Video Camera and Monitor Colony Counter Controlled Rate Freezer Centrifugal Elutriator Flow cytometer .

` ` ` ` ` Pippetes Culture Vessels Sterile Containers Syringes and Needles Sterilization Filters .

` Contamination by microorganism remains a major problem in tissue culture bacteria. can be widespread and wipe out your entire stock . yeast. mycoplasma. and fungal spores ` Contaminations can be minor and confined to one or two cultures and infect a whole experiment.

` Catastrophes can be minimized if: 1. media. etc are not shared with other people or use for different cell line. Reagents are checked for sterility before use. Bottles. preferably by phase contrast 2. The standard of sterile technique is kept high all the time . 5. Culture are checked carefully by eye and on a microscope. 4. Cultures are maintained without antibiotic at least part of the time to reveal cryptic contaminations. 3.

invisible under regular microscopy. presents one of the major threats Undetected. it can spread to other cultures around the laboratory.` Mycoplasmal infection. Essential to back up visual checks with mycoplasma test ` ` .

` ` ` ` ` Quite Area Work Surface Personal Hygiene Reagents and media Culture .

` ` ` ` ` ` Swabbing Capping Flaming Handling Bottles and Flasks Pippeting Pouring .

. not recirculated .most stable airflow and best sterile protection to the culture and reagents . paralel to the work surface.the airflow blows from the side facing you.` Horizontal.

` Vertical.more protection to the operator .air blows down from the top of the hood onto work surface and is drawn through the work surface and either recirculated or vented . .

` Class II vertical flow biohazard hood should be used if potentially hazardous material is being handled human or primate derived cultures. virally infected cultures. ` . etc ` HEPA filters above the work surface should be monitored for airflow and holes UV lights are used to sterilize the air and expose work surfaces in laminar-flow hoods between uses.

and B3) is designed to protect personnel. B2. ` . B1. product and enviroment.` BSC Class I is designed to protect the product BSC Class II (Types A.

Risk Assesssment is an important principle that has become incoporated into most modern safety legislation.` ` ` Cell culture laboratory has a number of particular risks associated with culture work. Standard Operating Procedures (SOP) if the procedure is deemed to carry any significant risk beyond the commonplace then a SOP should be defined .

` Safety regulation . CDC. .Biosafety in Microbiological and Biomedical Laboratories (BMBL).Occupational Health and Safety Administration (OSHA) .

Determine that the individual is already trained Equipment A general supervisor should be appointed to be in charge of all equipment maintenance. .` General Safety Operator It is responsibility of the institution to provide the correct training.

powerful solvent and skin penetrant. cytotoxic drugs Gases CO2. O2. Mutagens. carry many substances through the skin. N2 are not harmful in small amounts .Glassware and sharp items The common form of injury in tissue culture results from accidental handling of broken glass and syringes needles Chemical toxicity DMSO. carcinogens.

asphyxiation and explosion Fire Associated with tissue culture stem from the use bunsen burners for flaming. together with alcohol for swabbing and sterilization. .Liquid Nitrogen Three major risks are associated with liquid nitrogen: frostbite.

High energy sources x rays macchines.Labeled reagents Irradiation from higher energy emmiters (32P. 51Cr) and . 125I.Ingestion Radiolabeled compounds can be ingested by being splashed on the hands or via aerosol generated by pipetting or the use of syringe . UV sources .` Radiation . 131I.

Transport specimen in double wrap container .` Biohazards .Carry out dissection and subsequent culture in a designated Class II biohazard hood. and work .Human biopsy material .Genetic Manipulation .Enter all specimens into logbook on receipt.Disposal ` Sample should be handled with caution . place the specimens in a secure refrigerator marked with biohazard label .

instruments. into disinfectant or an autoclave etc.Discard all glassware. . pippetes. - Put all cultures in a plastic box with tape or labels identifyng the cultures as biohazardous and with the name of person responsible and the date on them ..- Avoid the use of sharp instruments in handling specimens.

Become familiar with proposed work activities (procedures.` Enables the professional (biosafety officer. personnel) Be knowledgeable and credible partner with the investigator to develop a safe and secure environment for the work . equipment. responsible official) to: .

` Review all activities associated with infectious materials .Proposed work activities Personnel Storage Transfer and transport Destruction .

` Safety risk group (RG) RG 1 : No or low individual and community risk Unlike to cause human or animal diseases RG 2 : Moderate individual risk. but effective treatment and preventive measure are available and risk of spread of infection is limited. low community risk Can cause diseases but unlikely to be a serious hazard. Lab exposure may cause serious infection. .

` RG 3 : High individual risk. usually causes serious human or animal diseases but does not ordinarily spread. Usually causes serious human or animal diseases and can be readily transmitted. Effective treatment and preventive measure are not usually available . Effective treatment and preventive measures are available RG 4 : High individual and community risk. low community risk.

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