OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number Initial Issue date
STGL. SWP.77.1 18/04/2010
School/ Divisional Unit
STGL
Current version
Current Version
1
Issue date:
18/04/2010
Next review date
18/04/2012
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 Assay
Description:
Associated risk assessment title and location: STGCL.RA.77.1
Describe the activity or process
This SWP describes methodology for crystal violet staining in clonogenic assay. Clonogenic assay or
colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a
colony. The colony is defined to consist of at least 50 cells. The dye in this assay, crystal violet, stains
DNA. So colonies formed can be visualised and counted after staining.
The hazards associated with this procedure are according to the specific compound used, and also the
vehicle in which that compound is dissolved. It is imperative that before carrying out an assay, the
individual reads all available MSDSs.
Procedure:
Optimisation of cell seeding density
The cell density required for the cells to grow within 3 weeks must be determined. This involves seeding
plates with a range of different cell numbers and allowing them to grow for 3 weeks. Usually, the cell
number range is between 100-600 cells per well depending on the doubling time of the cell line.
Preparation of Crystal violet stain
0.5g of crystal violet is weighed and dissolved in 20ml of methanol and made up to 100ml with milli-Q
water. (NB: crystal violet and methanol are hazardous substances).
PBS
1 sashe of Phosphate buffered saline tablets from In Vitrogen was dissolved in milli-Q water to make 1L of
PBS. The PBS was filtered using 0.2 um filter. (For filtration details see preparation of media SWP---).
Procedure
1. Culture the cell line to be tested until the required cell number is reached (1 x 75cm 2 flask is enough)
2. Detach the cells with 0.5% trypsin and determine the cell concentration with a haemocytometer.
3. Label 6-well plates with the appropriate cell density, making sure that duplicates or triplicates are
added for each cell number.
4. Add 3mls of fresh medium into each well.
5. Dilute the samples down to a concentration of 103 cells/ml with fresh medium.
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Date Effective: 01/01/2007
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Describe the activity or process
6. Aliquot the corresponding number of cells into each well (0.1ml for 100 cells……….0.6mls for 600
cells, etc).
7. Allow the cells to grow and change the culture medium every 3 days.
8. Monitor the cell growth and when the number of cells in a colony has reached 50 or more, the cells
can be stained and counted. Different cell-lines have different doubling times so you have to take into
account the doubling time for that cell-line and monitor the growth according to that. (Take care when
aspirating medium from wells; take note whether cell type is strongly adherent as they might dislodge
easily).
9. Culture Using a pipette, aspirate the cell-culture medium from the 6-well plates (ensure that cells are
strongly adherent to bottom of well).
Crystal Violet staining Procedure:
1. Using a pipette, aspirate the cell-culture medium from the 6-well plates (ensure that cells are strongly
adherent to bottom of well). For non-adherent cells, centrifuge cell-culture medium in micro-titre plate
holder at 1500rpm for 5min.
2. Using a pipette, add 2mls of PBS into each well for washing purposes. Dispose the medium
appropriately in a waste beaker.
3. Add 1ml of crystal violet solution into each well and incubate for 10mins at room temperature (Crystal
violet is a hazardous substance).
4. Wash each well with a gentle stream of water under a running tap water.
5. Invert the plates and allow to dry.
6. Count the number of ‘darkly’ stained colonies (greater than or equal to 50) under the white light or
phase-contrast microscope at low magnification
Plating efficiency (PE) calculations:
PE = (no. of colonies) / (no. of original cells seeded)
List all resources required including
plant, chemicals, personal protective
clothing and equipment, etc
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Crystal violet (NB: This is classified as Hazardous)
Methanol (NB: This is classified as Flammable and Toxic)
PBS (Phosphate buffered saline)
Distilled water
PPE – lab-gown, gloves, safety glasses with side shields
Waste-beaker
List potential hazards and risk controls
including specific precautions required
Crystal Violet
 Inhaling of dust is irritating to mucous membrane and upper respiratory tract. Wear a Class P1
(Particulate) respirator. Avoid inhaling the dust and if inhaled remove to fresh air. If breathing
becomes difficult. See physician.
 Contact with skin can be harmful. Wear gloves and lab-gown. In case of contact with skin,
immediately wash with soap and copious amounts of water
 There is a risk of serious damage to eyes. Wear dust proof goggles or goggles with side-shields.
In case of contact with eyes, flush with copious amounts of water for at least 15minutes. Assure
adequate flushing by separating the eye-lids with fingers. See physician
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Date Effective: 01/01/2007
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List potential hazards and risk controls
including specific precautions required

Ingestion can be harmful. If ingested rinse mouth with water. If swallowed, wash out mouth with
water, provided person is conscious. Contact Poison Information Centre on 13 11 26. Do not
induce vomiting.
 It emits toxic fumes under fire conditions. Wear self contained breathing apparatus and protective
clothing to prevent contact with eyes and skin.
 Crystal violet is very toxic to aquatic organisms. It may cause long term adverse affects in the
aquatic environments. So dispose the waste in the waste container.
Methanol
 Highly flammable, emits toxic fumes under fire conditions. Vapor may travel to considerable
distances to source of ignition and flash back. Container explosion may occur under fire
conditions. Keep away from all the ignition sources in the flammable cabinet. Do not handle near
open flames or sources of sparks.
 Toxic, danger of very serious irreversible effects by inhalation, in contact with skin and if
swallowed. Wear PPE. Follow the procedures under Crystal violet if it gets into eyes or skin or
ingested.
List emergency shutdown instructions
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Spillage: Contact emergency services where appropriate, use PPE equipment , clear area of all
unprotected personnel, prevent spills entering drains or waterways. Contain spillage , then cover /
absorb spill with non-combustible absorbant material (vermiculite or sand), collect and place in
suitable containers for disposal.
Fire and explosion: Highly flammable. Toxic gases may be evolved under fire conditions.
Eliminate all ignition sources. Evacuate area and contact emergency services (X666). Notify
others of hazard. Wear full PPE including self contained breathing apparatus and protective
clothing to prevent contact with eyes and skin. Use water fog to cool intact containers and nearby
storage areas.
Extinguishing: Dry agent, carbon-dioxide or foam. Prevent contamination of drains or
waterways.
List clean up and waste disposal
requirements
Crystal violet waste should be collected in a labelled waste bottle, indicating the concentrations of each
chemical ready to be collected by the UNSW waste pick-up and disposal service.
List legislation, standards and codes of
practice used in the development of the
SWP
Supervisory approval, training, and review
Supervisor: Dr Mohammad Pourgholami
Signature:
Plant custodian: Dr Mohammad Pourgholami
Signature
List competency required – qualifications, certificates, licencing, training - eg course or
instruction: Training in this SWP & familiarity with relevant MSDS, Hazardous Substance
Training
SWP review date: 18/04/2012
Responsibility for SWP review: Samina Badar
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
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Page 4 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007