Aseptic Technique Chapter 6

Objectives of aseptic technique Contamination must be excluded Cultures are checked carefully by eye via a microscope Cultures are maintained without antibiotics Reagents are checked for sterility before use Bottles of media or other reagents must not be shared with other people or used for different cell lines Maintenance of high sterile techniques

Elements of Aseptic Environment Work surface Clear the surface of the hood Swab the surface with 70% alcohol Keep items related to working experiment Swab between procedures

Elements of Aseptic Environment Work Surface Keep a clear central working space/area Pipette should not be contaminated Mob any spillage and swab with 70 % ethanol After experiment – swab again

Absolutely no talking while performing bench work to prevent ur saliva from falling into culture

Elements of Aseptic Environment Personal Hygiene Hand washing removes microorganisms and dead skin Surgical gloves may be worn Caps, gowns and face masks Tie back long hair Talking is permissible with a barrier between you and culture Vertical 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

Elements of Aseptic Environment Reagents and media Undergo strict quality control by companies Outside surface of bottles might not be sterile Bottles wrapped in polyethylene Wrappings removed before use in hood Unwrapped bottles swabbed by 70 % ethanol Polyethylene 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

Elements of Aseptic Environment Cultures Imported ones – contamination at source or transit Should be quarantined Kept away or incorporated into main stock Antibiotics usage – suppress and not eliminate contamination Should be quarantined for contamination and kept separately from rest of your stocks.

Elements of Sterile Handling Swabbing Swab work surface with 70% alcohol Clean spillage Swab bottles – cold storage/water bath/incubators Label with alcohol resistant markers

Elements of Sterile Handling Capping Deep screw caps preferred to stoppers No detergent remains in rubber liners of caps Screw cap covered with aluminum foil to protect neck of bottle from deposition of dust

Elements of Sterile Handling Flaming Open bench – flame glass pipettes + neck of bottles and screw caps Place open side down on clean surface and flamed before being replaced Flaming not advisable in hoods Screw caps should be flamed while opening and closing bottles. Screw caps must be held in hands during pipetting to avoid flaming or laying them down Flaming 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

Elements of Sterile Handling Handling bottles and flasks Open bench – Do not leave open vertical bottles to avoid spillage Bottle racks – keep bottles tilted Hoods – Leave bottles vertical and open Hoods – No blockage between open vessel or sterile pipette and HEPA air filter

Elements of Sterile Handling Pipetting Pipettes of sizes 1 ml, 5 ml, 10 ml, 25 ml and 100 ml is available Unwrapping pipettes should be done carefully Glass pipettes must be sterilized before use in hood Mouth pipetting should be strictly avoided Glass 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.

Laminar Flow Hood Horizontal Hood Airflow blows from the side facing you, parallel to work surface No recirculation of air Stable airflow and best sterile protection to culture and reagents

Laminar Flow Hood Vertical Hood Air blows down from top of hood onto work surface. Drawn through work surface and either recirculated or vented Protection to operator Avoids overspill in work area

Different types of hoods Class I for simple and non-pathogenic Class II for potentially hazardous material (human or primate-derived cultures, virally infected cultures etc) Class III for known human pathogens

Cytotoxicity hood Protection against chemical and radiochemical hazards Carbon filter trap in recirculating airflow or hood with all effluent vented to outside the building

Pressure and Air flow in a hood Pressure drop – Manometer Air flow – Anemometer Below 0.4 m/s (80 ft/min) – stability of airflow is lost – sterility cannot be maintained If resistance pressure drops in hood then airflow rate also drops

Routine maintenance checks of hoods Primary filters (3-6 months) – removed Horizontal-flow hoods – removal – discarded or washed in soap and water Vertical- flow hoods – Biohazard hoods are internal – only an engineer can replace - Incinerated or autoclaved and discarded

Routine maintenance checks of hoods HEPA filter – once every 6 months Should be monitored for airflow and holes Monitoring done by engineers Biohazard cabinets – bagging and disposing of filters by incineration

Routine maintenance checks of hoods Weekly checks – spillage cleaned, sterilized with 5 % phenolic disinfectant and 70 % ethanol Do not let any material block the airflow. Check regularly for any droppings Labs keep hoods running to keep area clean Ultraviolet light sterilizes but do not reach crevices – alcohol by capillary action

Incubators Major source of contamination Should be cleaned regularly (weekly or monthly) Washing racks or shelves by nontoxic detergent – Decon or Roccall Traces of detergent removed by ethanol- before placing back shelves

Incubators Fungicide – 2 % Roccall or 1 % Copper Sulfate placed in humidifier tray Micropore filtration and laminar airflow to inhibit circulation of microorganisms Fungicide is not effective as it does not touch all surfaces

Boxed Cultures Repeated contaminations in incubator Use sandwich boxes Enclose dishes, plates and flasks with slackened caps Swab 70% ethanol

Gassing with CO2 This 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-15-162-06-60). 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; and against 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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