OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM.SWP006
Initial Issue date
23rd June 2009
School/ Divisional Unit
School of Medical Sciences
Oncology Research Unit/Neuromuscular and
Regenerative Medicine Unit
Current version
Current Version
Next review date
1.0
23rd June 2012
Issue date
23rd June 2009
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
form.
Safe Work Procedure Title and basic description
Title: Clonogenic-colony suppression assay
Description: Procedure for measuring a compound/proteins ability to suppress colony formation in mammalian
cell lines.
Associated risk assessment title and location: 06_Colony Suppression Assay_JS
Describe the activity or process
Introduction to the procedure:
The clonogenic survival assay is the most commonly used assay to assess growth inhibition in vitro. Cultured cells are
seeded into tissue culture dishes at low densities to allow the formation of individual clonogenic colonies. In this
circumstance this method is used to determine the effect on cell growth of either knocking down a protein of
interest (siRNA) or in the presence of cytotoxic drugs.
Procedure:
SETTING UP ASSAY PLATES
FOR CYTOTOXICITY ASSAYS
1. Mammalian cell lines are maintained in T75 tissue culture flasks
2. Prior to use cells are trypsinized and counted using a hemocytometer.
3. For a 6-well plate cells are seeded at ~ 500 cells/well/2ml for a 12 well plate cells are seeded at 100
cells/well/0.5ml (this needs to be optimized for each cell line being used).
4. 24hrs post plating cells are incubated in media containing increasing concentrations of drug/ compound and a
well for a vehicle control. Drug dose is specific to the compound being used and needs to be optimized for each
compound. Cells are treated for 72hrs with cytotoxic drug. Drug containing medium is then removed and is
replaced with fresh culture media. Media is then changed every 3days for seven to ten days until visible colonies
form.
FOR siRNA
1.
Mammalian cell lines are maintained in T75 tissue culture flasks and plated into 6-well culture dishes at a
density of 1.0X105 cells /well/2mls 24hrs prior to siRNA transfection.
2. Cells are transfected with siRNA (as described in the ORU_NRMU_siRNA transfection SWP).
3.
72hrs post transfection cells are harvested and reseeded into 6-well plates at ~ 500 cells/well/2ml for a 12 well
plate cells are seeded at 100 cells/well/0.5ml (this needs to be optimized for each cell line being used).
4.
Media is changed every 3days for seven to ten days until visible colonies form.
STAINING CELLS FOR ANALYSIS
1. After 10-15 days media is removed and colonies washed twice with 1XPBS.
2. Colonies are then fixed for 15 min at RT with 10% Acetic acid and 10% methanol.
3. Colonies are stained with 0.4% Crystal violet and 20% Ethanol at RT for 40 mins.
4. Staining solution is then removed and plates are washed once with distilled water and air dried. Plates can then
be photographed or scanned to visualize and count colony numbers.
Surviving fraction can then be determined as follows: colony number/ (number of cells seeded X plating efficiency).
Plating efficiency is determined by the colony number divided by the number of cells seeded at the beginning of
experiment (control condition).
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Page 1 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Chemicals/buffers:
Fixing solution
10 mls Acetic acid
10mls 100% Methanol
80mls dH20
Staining solution
0.4g Crystal violet
20mls 100% Ethanol
80mls dH20
1 X DMEM complete media
1 X DMEM media
10% FBS
Equipment:
Sterile 6-well tissue culture dish, 12 well tissue culture dishes, Sterile pipettes (5,10 and 25ml), Pipette gun, Pipettes and pipette tips
Large equipment
Tissue culture biohazard hood, Tissue culture incubators, 37 water bath, 4C fridge, dissecting microscope/scanner.
PPE: Latex Gloves
Gown
Safety Goggles
Enclosed shoes
List potential hazards and risk controls including
specific precautions required
Chemicals and reagents:
Crystal violet: Hazardous in case of eye contact (irritant), of ingestion, of inhalation. Slightly hazardous in case of skin
contact (irritant). PPE to be worn.
Methanol: Highly flammable, keep away from naked flames and ignition sources. Toxic by inhalation, in contact with
skin and if swallowed. Methanol used for the preparation of fixation buffer is decanted within the laboratory in a
well ventilated area. PPE to be worn.
Ethanol: Highly Flammable, keep away from flames and ignition sources. Irritating to eyes. Repeated exposure may
cause skin dryness. Vapours may cause headaches, nausea and vomiting. Use in a well ventilated area and PPE to be
worn.
Acetic acid: Very hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation.
Hazardous in case of skin contact (corrosive, permeator), of eye contact (corrosive). Liquid or spray mist may
produce tissue damage particularly on mucous membranes of eyes, mouth and respiratory tract. Skin contact may
produce burns. Inhalation of the spray mist may produce severe irritation of respiratory tract, characterized by
coughing, choking, or shortness of breath. Decanting acetic acid must be done in a fume hood wearing PPE.
List emergency shutdown instructions
Methanol: Do not turn on, or extinguish any ignition source - no smoking. Take precautionary measures against
static discharges. Use dry chemical or foam to extinguish if there is a naked flame.
Ethanol: Do not turn on, or extinguish any ignition source - no smoking. Take precautionary measures against static
discharges. Use dry chemical or foam to extinguish if there is a naked flame.
List clean up and waste disposal requirements
Crystal violet: Disposal of general crystal violet waste is in a liquid tissue culture biohazard waste container located
in the laboratory. For spills: Use appropriate tools to put the spilled solid in a biohazard waste disposal container.
Finish cleaning by spreading water on the contaminated surface and dispose of in appropriate biohazard waste
container.
Methanol: Used methanol is discarded in marked liquid biohazard waste containers within the laboratory. For spills:
Contain spills as much as possible. Liquid spills should be absorbed prior to disposal. (“Silicate” type absorbent
materials are suggested). Minimise dilution water to control spill volume.
Ethanol: Used ethanol is discarded in the general laboratory liquid biohazard containers. For spills: Contain spills for
salvage or disposal. Liquid spills should be absorbed prior to disposal. (“Silicate” type absorbent materials are
suggested). Minimise dilution water to control spill volume.
Acetic acid: Used acetic acid is disposed of in a marked acid waste biohazard waste container in the laboratory. For
spills: Dilute with water and mop up, or absorb with an inert dry material and place in an appropriate general
laboratory waste disposal container. If necessary: Neutralize the residue with a dilute solution of sodium carbonate.
In the event of an emergency people to contact include:
Fire Wardens: Dr Justine Stehn and Dr Anthony Kee
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
First Aid: Renee Szokoli
List legislation, standards and codes of practice
used in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3: 2006 Safety in laboratories Part 3: Microbiological aspects and containment facilities
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2243.3:2006 Safety in Laboratories Part 7 Electrical aspects
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
Supervisory approval, training, and review
Supervisor: Peter Gunning
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction to Lab, Training in this SWP
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Page 3 of 3
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007