2 Objectives of aseptic technique Contamination must be excludedCultures are checked carefully by eye via a microscopeCultures are maintained without antibioticsReagents are checked for sterility before useBottles of media or other reagents must not be shared with other people or used for different cell linesMaintenance of high sterile techniques
3 Elements of Aseptic Environment Work surfaceClear the surface of the hoodSwab the surface with 70% alcoholKeep items related to working experimentSwab between procedures
5 Elements of Aseptic Environment Work SurfaceKeep a clear central working space/areaPipette should not be contaminatedMob any spillage and swab with 70 % ethanolAfter experiment – swab again
6 Absolutely no talking while performing bench work to prevent ur saliva from falling into culture
7 Elements of Aseptic Environment Personal HygieneHand washing removes microorganisms and dead skinSurgical gloves may be wornCaps, gowns and face masksTie back long hairTalking is permissible with a barrier between you and cultureVertical laminar hood has a barrier between you and culture – can talk but keep limited talking. If you have a cold then wear a mask to prevent spread of infection
8 Elements of Aseptic Environment Reagents and mediaUndergo strict quality control by companiesOutside surface of bottles might not be sterileBottles wrapped in polyethyleneWrappings removed before use in hoodUnwrapped bottles swabbed by 70 % ethanolPolyethylene wrapped bottles keeps them clean and allows them to be placed in water bath to be warmed or thawed. Unwrapped bottles should be swabbed with ethanol after their removal from refrigerator or water baths
9 Elements of Aseptic Environment CulturesImported ones – contamination at source or transitShould be quarantinedKept away or incorporated into main stockAntibiotics usage – suppress and not eliminate contaminationShould be quarantined for contamination and kept separately from rest of your stocks.
10 Elements of Sterile Handling SwabbingSwab work surface with 70% alcoholClean spillageSwab bottles – cold storage/water bath/incubatorsLabel with alcohol resistant markers
11 Elements of Sterile Handling CappingDeep screw caps preferred to stoppersNo detergent remains in rubber liners of capsScrew cap covered with aluminum foil to protect neck of bottle from deposition of dust
12 Elements of Sterile Handling FlamingOpen bench – flame glass pipettes + neck of bottles and screw capsPlace open side down on clean surface and flamed before being replacedFlaming not advisable in hoodsScrew caps should be flamed while opening and closing bottles. Screw caps must be held in hands during pipetting to avoid flaming or laying them downFlaming will disrupt laminar flow (sterility of hood and its containment of biohazardous material). Open flames can damage high efficiency particulate air (HEPA) filter or melt some plastic interior fittings
13 Elements of Sterile Handling Handling bottles and flasksOpen bench – Do not leave open vertical bottles to avoid spillageBottle racks – keep bottles tiltedHoods – Leave bottles vertical and openHoods – No blockage between open vessel or sterile pipette and HEPA air filter
14 Elements of Sterile Handling PipettingPipettes of sizes 1 ml, 5 ml, 10 ml, 25 ml and 100 ml is availableUnwrapping pipettes should be done carefullyGlass pipettes must be sterilized before use in hoodMouth pipetting should be strictly avoidedGlass pipettes – insert cotton plug in top of glass pipette before sterilization to keep pipette sterile. The plug prevents contamination from bulb or pipetting aid and cross-contamination from pipette to bulb or pipetting aid. If plug becomes wet, discard the pipette into disinfectant for return to wash up. Plugging pipettes for sterile use and also their removal before washing is a tedious job. The disposable plastic ones come plugged. It is advisable to use individually wrapped pipettes as they are free from contamination rather than buying all plastic ones in a common packing which would lose the purpose of sterility if opened. They are free of chemical and microbial contamination and reduce washing requirements.
16 Laminar Flow Hood Horizontal Hood Airflow blows from the side facing you, parallel to work surfaceNo recirculation of airStable airflow and best sterile protection to culture and reagents
17 Laminar Flow Hood Vertical Hood Air blows down from top of hood onto work surface.Drawn through work surface and either recirculated or ventedProtection to operatorAvoids overspill in work area
18 Different types of hoods Class I for simple and non-pathogenicClass II for potentially hazardous material (human or primate-derived cultures, virally infected cultures etc)Class III for known human pathogens
19 Cytotoxicity hoodProtection against chemical and radiochemical hazardsCarbon filter trap in recirculating airflow or hood with all effluent vented to outside the building
20 Pressure and Air flow in a hood Pressure drop – ManometerAir flow – AnemometerBelow 0.4 m/s (80 ft/min) – stability of airflow is lost – sterility cannot be maintainedIf resistance pressure drops in hood then airflow rate also drops
21 Routine maintenance checks of hoods Primary filters (3-6 months) – removedHorizontal-flow hoods – removal – discarded or washed in soap and waterVertical- flow hoods – Biohazard hoods are internal – only an engineer can replace- Incinerated or autoclaved and discarded
22 Routine maintenance checks of hoods HEPA filter – once every 6 monthsShould be monitored for airflow and holesMonitoring done by engineersBiohazard cabinets – bagging and disposing of filters by incineration
23 Routine maintenance checks of hoods Weekly checks – spillage cleaned, sterilized with 5 % phenolic disinfectant and 70 % ethanolDo not let any material block the airflow. Check regularly for any droppingsLabs keep hoods running to keep area cleanUltraviolet light sterilizes but do not reach crevices – alcohol by capillary action
24 Incubators Major source of contamination Should be cleaned regularly (weekly or monthly)Washing racks or shelves by nontoxic detergent – Decon or RoccallTraces of detergent removed by ethanol- before placing back shelves
25 IncubatorsFungicide – 2 % Roccall or 1 % Copper Sulfate placed in humidifier trayMicropore filtration and laminar airflow to inhibit circulation of microorganismsFungicide is not effective as it does not touch all surfaces
26 Boxed Cultures Repeated contaminations in incubator Use sandwich boxes Enclose dishes, plates and flasks with slackened capsSwab 70% ethanol
27 Gassing with CO2This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB ). NCC is an equal opportunity employer and does not discriminate on the following basis:against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; andagainst any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.
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