OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM.SWP037
School/ Divisional Unit
School of Medical Sciences
Initial Issue date
26 June 2009
Current version
1.0
Current Version
Issue date
26 June 2009
Next review date
26 June 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: MTT Viability Assay
Description: To perform a cell proliferation or cytotoxicity assay in any mammalian cell line using MTT
Associated risk assessment title and location: 37_RA_MTT assay_TB
Describe the activity or process
1.
Prepare MTT stock concentration of 2mg/ml from powder in PBS. Filter sterilize and store at 4°C.
2.
Plate mammalian cells at the required density in a 96 well plate (this will need to be optimized for each cell line),
and allow cells to adhere for 24hrs at 37°C and 5% CO2 before proceeding with assay.
2a. Optional*: Treat cells with cytotoxic compound of interest, diluted serially over an 8 log range of concentrations.
3.
Add 50μl MTT stock solution directly to the cells in culture medium and incubate at 37°C with 5% CO2 for 4hrs.
4.
Aspirate media from wells. Add 100μl DMSO to each well to solubilize the purple precipitate and mix solutions
thoroughly using a plate shaker.
5.
Measure the absorbance at 540nm for each well.
6.
Subtract the average absorbance values of the control group (media only) from the absorbance of the
experimental groups (6a. vehicle control group from the drug-treated groups).
7.
Determine cell viability by plotting absorbance versus log compound concentration. Higher absorbance intensity
values correlate to an increase in total metabolic activity from cells.
* See: SWP titled “handling cytotoxic compounds”
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Reagents and equipment:

MTT (Thiazolyl Blue Tetrazolium Bromide; Sigma cat. # M2128) labeling reagent

DMSO (Dimethyl sulfoxide)

PBS

96 well tissue culture plate

mammalian cells in appropriate medium

Tissue culture incubator (37°C, 5% CO2 incubator)

Microplate reader with 540 and 690nm filters

digital multi-channel pipette
Personal Protective Clothing

Lab gown

gloves
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Page 1 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List potential hazards and risk controls including
specific precautions required



All biological material manipulation must be conducted in a biological safety cabinet.
MTT may be harmful if inhaled, absorbed through the skin or ingested. Use protective gloves, safety glasses and
lab gown when handling MTT. If product comes into contact with skin or eyes wash off immediately with plenty of
water. If inhaled, move person into fresh air.
DMSO is slightly hazardous in case of inhalation (lung irritant), skin contact, eye contact and ingestion. Use
protective gloves, safety glasses and lab gown when handling DMSO.
Eye contact: immediately flush eyes with running water for 15 minutes
Skin contact: wash with soap and water
Inhalation: if inhaled, remove to fresh air. If not breathing, give artificial respiration
Ingestion: do NOT induce vomiting unless directed to do so by medical personnel
List emergency shutdown instructions


Chemical spill: In the case of a chemical spill notify a person immediately and access the chemical spills kit
located in Rm502.
Electrical fault: Refer to SWP “tissue culture incubator” and “microplate reader” for correct shutdown procedures.
List clean up and waste disposal requirements



Ethanol and wipe-down all used surfaces.
Add bleach to plates prior to discarding them in biological waste containment. Where plates contain cytotoxic
compounds refer to SWP “handling cytotoxic compounds” for correct disposal procedures.
PC2 Safe Work Practices apply. Wash hands before leaving the PC2 facility.
List legislation, standards and codes of practice used
in the development of the SWP










NSW OHS Act 2000
NSW OHS Regulation 2001
Safe Work Procedure Form (OHS026)
Australia Dangerous Goods Code.
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
Australian Standard AS2243.3-2002. Safety in laboratories. Part 3: Microbiological aspects and containment
facilities.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
Children’s Cancer Institute Australia Safe Operating Procedures
Safe Work Procedure Form (OHS026)
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 the Lab, Training in this SWP.
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Page 2 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007