Safe Work Procedure - Tissue Culture Equipment

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--Mark Hill 18:23, 11 March 2010 (EST) This SWP should really be split into 3 separate parts Biosafety cabinets, incubators and waterbaths. Also TL should have separate SWPs. Does not refer to CO2 cylinder handling


Faculty/Division: Medicine

School/ Divisional Unit: School of Medical Sciences

Document number:

Initial Issue date: 18.6.09

Current Version: 1

Current Version Issue date: 18.6.09

Next review date: 18.6.10

The Safe Work Procedure - Guide to Writing (OHS027) should be consulted to assist in the completion of this form.

Contents

Safe Work Procedure Title and basic description

Title: Use of Tissue Culture equipment (Biosafety cabinets, incubators and waterbaths)

Description: To ensure that the equipment used to perform tissue culture is used effectively in order to achieve culture sterility, biosafety, OGTR and OHS.

Associated risk assessment title and location: Risk Assessment - Tissue Culture Equipment

Describe the activity or process

  1. Biosafety Cabinet- is the primary barrier protection for individuals working with biohazardous materials. Laboratory procedures that could create airborne biohazards should always be performed in a biosafety cabinet as it protects laboratory workers and the environment from aerosols or droplets that could spread biohazardous material. Biosafety cabinets must undergo an annual certification by an outside contractor to ensure that the cabinet is working according to the Safety Standards.
  2. Incubator- is utilized to maintain the culturing of primary cells and cell lines at 37oC and 5% CO2.
  3. Waterbath- is utilized to warm solutions to 37oC which is the preferred temperature used for the culturing of primary and cell lines.

As for all the equipment described above the date on the electrical safety tags must be checked, do not use if out of date and report to Laboratory manager.

Description of activities:

1. Biosafety Cabinet

  • Wash hands before starting any work.
  • Personal Protective gear must be worn; these include back opening, long sleeved laboratory gown and gloves. Use the gowns dedicated for the tissue culture room only. Outside general laboratory gowns must not be used inside the room. The cuffs of the gloves should be pulled up and over the cuffs of the coat sleeves.
  • To UV the biosafety cabinet firstly ensure that the front glass access panel is fully down and in place. The UV light source is turned on by holding the UV switch on for 3 secs. The UV light only needs to be on for a maximum of 15-20 minutes before use to have a germicidal effect. The UV light will turn off automatically after 60 mins. Do not lift the glass window to look directly inside the cabinet whilst the UV indicator light is illuminated.
  • To switch cabinet on press On button until a beep is heard, press and hold the up pointing arrow to elevate the glass window, turn on the light. Operate cabinet blowers for five minutes before beginning work to allow the cabinet to purge or remove particulates from the cabinet.
  • Disinfect the work area. Wipe the work surface, interior walls and surface of the window with 70% ethanol. Introduce only those items that are required to perform the procedures. Each item should be wiped with 70% ethanol prior to placing it into the cabinet in order to reduce the introduction of contaminants.
  • Do not block the front grille with papers, equipment, etc., as this may cause air to enter the work space area instead of being drawn through the front grille and into the HEPA filter. Arms should be raised slightly and operations should be performed on the work surface at least four inches from the front grille. The middle third area is ideal.
  • If culture media or other fluids need to be aspirated, suction or aspirator flasks should be connected to an overflow collection flask containing disinfectant.
  • When work is completed remove the items that you have introduced. The interior surfaces of the cabinet should be cleaned with 70% ethanol.
  • Switch cabinet off by pressing the down pointing arrow until the glass window is fully down, press the ON button and hold for 5 full seconds, cabinet will be switch off.
  • Investigators should remove their gowns and gloves and thoroughly wash their hands before exiting the laboratory.

2. Incubator

  • Each researcher is responsible for the incubator their cells are kept in. Ensure they are kept cleaned and water trays in CO2 incubators are filled with distilled water.
  • All material placed in the incubator should be clearly labeled with the name and date.
  • All material should be removed after the appropriate time of incubation to prevent overcrowding or contamination of the incubator.
  • Any spillage should be reported immediately to the person in charge of the room and should be cleaned up in a manner appropriate to the material spilled.
  • Check temperature and CO2 levels for incubator and water levels daily.
  • All biological spills on trays and shelves must be wiped up immediately.
  • Incubators should be cleaned at least every 2-3 months
  • It is the responsibility of the researcher to check that CO2 gas cylinders supplying CO2 to their incubators are sufficiently full.
  • Only trained staff are to change gas cylinders.

3. Waterbath

  • Ensure that the water is always clean hence the waterbath must be clean every month.
  • To clean waterbath ensure that power supply to the instrument is switched “off”.
  • Remove the water and wipe the inside with 70% ethanol
  • Add enough MilliQ water to cover the base tray. An antifungal agent can be added to the water alternatively a couple of copper coins.
  • Ensure that the instrument is properly connected to power supply, switch “on”
  • To clean waterbath ensure that power supply to the instrument is switched “off”
  • Remove the water but scooping as much as possible with a plastic beaker and then gently tilting the waterbath over the sink
  • Wipe the inside with 70% ethanol
  • Add enough distilled water to cover the base tray. An antifungal agent can be added to the water
  • Set the thermostat knob to 37oC.
  • Insert a thermometer in the water and when the correct temperature is reached the instrument is ready for use. DO NOT USE A MERCURY THERMOMETER.
  • The waterbath should remain on at all times
  • Report any discrepancy observed during operation of the instrument to the person in charge of the room.

List all resources required including plant, chemicals, personal protective clothing and equipment, etc

(changed from original --Mark Hill 18:31, 11 March 2010 (EST))

  • Always wear long sleeve lab gowns, latex gloves and eye protection when working in the lab
  • Ear Protection
  • Biological Safety Cabinet
  • 70% ethanol
  • Sonicator
  • Kim-wipes

List potential hazards and risk controls including specific precautions required

(this requires more details - --Mark Hill 18:31, 11 March 2010 (EST))

1. Potential Hazards

  • Exposed to UV radiation to skin and eyes
  • Electrical fault

2. Risk Controls

  • All staff and students are trained to use the specific equipment
  • Laboratory induction and regular OHS discussions at Lab Meetings to reinforce the potential hazards

List emergency shutdown instructions

  • All items of equipment have “OFF” switches that can be used in emergency or
  • the emergency power shutdown switch located in the main lab and in the TC room

List clean up and waste disposal requirements

(more details required --Mark Hill 18:33, 11 March 2010 (EST))

Report any faults to the respective Laboratory Managers – (BioSafety Cabinets to ORU/MDU)

List legislation, standards and codes of practice used in the development of the SWP

--Mark Hill 18:34, 11 March 2010 (EST) there are specific Australian standards for this equipment that I have added.

NSW OHS Act 2000

NSW OHS Regulation 2001

Safe Work Procedure Form (OHS026)

Australian Standard AS2252.2-1985. Class II biological safety cabinets.

Australian Standard AS2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.

Australian Standard AS2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects

Supervisory approval, training, and review

Supervisor:

Signature:

Plant custodian:

Signature:

List competency required – qualifications, certificates, licencing, training - eg course or instruction:


SWP review date:

Responsibility for SWP review:

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