Transcript
STANDARD OPERATING PROCEDURE Procedure: School/Department: SOP prepared by: Version:
Spread-plating with glass spreader and ethanol School of Molecular Bioscience Sue Ling Lim, Nick Coleman SMB032.3
Section 1 - Personal Protective Equipment 1. Lab coat or gown 2. Enclosed shoes 3. Long hair MUST be tied back 4. Gloves if appropriate (e.g. if risk group 2 microbes used) 5. Safety glasses/goggles if appropriate (e.g. risk group 2 microbes) Section 2 – Potential Hazards + Safety precautions 1. Main risk of this procedure is that the container of 100% ethanol can catch fire. To minimise this risk, ensure the following precautions: - ethanol container is fireproof (e.g. glass or metal, not plastic) - ethanol container is at least 30 cm away from Bunsen burner - ethanol container has a fireproof cover/lid available (e.g. glass or metal, not plastic), and that the lid is kept close by (in easy reach) to enable any fires in ethanol container to be extinguished - ethanol container is stable and hard to knock over. A low flat container (e.g. crystallisation dish) is essential. Do not use a tall narrow container (e.g. measuring cylinder) - you may like to practice putting out a fire in the ethanol container before you start the procedure so you know what to do in an emergency. To do this safely, put a small amount of ethanol (e.g. 10 ml) in the container, ensure it is in an open location away from all flammables, light the ethanol carefully (e.g. with a match dropped in) and then once fire has started, put the lid on. Ensure, that someone experienced is present when you do this. - it is very important to keep calm in the event of a fire in the ethanol container (or any fire). If you panic, you risk spilling the flaming ethanol, and making the situation much worse. Knowing the location of safety equipment, and practising putting out a small controlled fire can help with this. 2. Bunsen burner and/or fires in ethanol dish can also lead to other items catching alight, therefore ensure the space you are working in is clear of clutter and any flammable items 3. Long hair is a particular hazard near open flames, ensure this is tied back 4. Know the locations of the nearest fire blanket, fire extinguisher and fire exit 5. If you are not comfortable with the use of glass spreaders and ethanol, or if you want to minimise the risks of this procedure, it may be possible to instead use disposable sterile plastic spreaders. These do not need flaming and thus are much safer to use. Consult your supervisor. 6. Workers with pre-existing medical conditions (e.g. allergy, immunocompromised state, chemical sensitivity) and workers who are pregnant or expecting pregnancy must consult with their supervisor AND medical specialist AND the university's WHS services before performing this procedure. If there are any serious concerns expressed by any of these individuals, this task must not be performed. Section 3 – Procedure 1. Put on protective equipment (as above). Prepare work area by clearing anything not required for spread plating. Especially take care to remove flammable items (paper towel, solvents etc). 2. Only use the minimum required amount of ethanol required (e.g. 100 ml) – do not leave the whole 2.5 L ethanol bottle on the bench. 3. Swab down bench with 80%(v/v) ethanol. Lay out all equipment necessary (agar plates, spreader/s, ethanol dish and lid, Bunsen burner) 4. Ensure Bunsen burner is at least 30 cm away from ethanol and clear of any flammable objects. 5. Label all plates beforehand (type of medium, bacteria, date, your initials) 6. Light Bunsen burner and put it on the safety flame (orange). Never leave the flame unattended. 7. Aseptically apply the bacteria onto the agar plate with a pipette. 8. Dip the glass spreader into the 100% ethanol, then take it out. Allow excess ethanol to briefly Creation date: 25/10/2012 Last review date: 27/3/2015
Next review due: 27/3/2017
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drain (2-3 sec), then pass the spreader with the residual ethanol through the flame. Do not hold the spreader in the flame for > 1 second – this will heat the glass too much, and kill the bacteria. 9. Allow any ethanol to burn off (3-5 seconds). Keep the spreader near the flame at this stage and over a clear area of the bench/hood, not near the ethanol dish. It’s possible that one or two flaming drips of ethanol will fall from the spreader. Do not be alarmed by this – if your work area is clear, and you keep the lit spreader away from the ethanol container, this wont cause problems. 10. Wait another 5 seconds or so until spreader has cooled a little, then spread the bacteria over the surface of the agar, rotating the agar plate as you spread. 11. Once each plate is completed, replace the spreader in the ethanol container. 12. Once all the spread-plates are completed, turn off the flame, wipe down the surface with 80% ethanol, and dispose of any leftover bacteria appropriately (either autoclaving or in bleach). 13. The ethanol used for spread plating can be reused several times, or can be disposed of to the sink if small volume (~100 ml). In that case, rinse down with a few litres of cold water to minimise generation of ethanol vapours in the sink. Section 4 – Disposal / Spills / Incidents 1. If container of ethanol catches alight, STAY CALM and place the cover on top – this will very quickly starve the fire of oxygen and put it out. 2. If the fire escalates and increases in size (e.g. if you spill the flaming ethanol), immediately cover the fire with a fire blanket or put out with a fire extinguisher 3. If the fire becomes too large to easily extinguish with a fire blanket or extinguisher, pull the fire alarm or notify the fire warden to evacuate the building, alert everyone nearby to evacuate. 4. If you spill ethanol on the bench, turn off the flame immediately, and clean up the spill with paper towel. Put the ethanol-soaked towel in the fume hood to evaporate off the ethanol (e.g. overnight), then dispose in regular rubbish. 5. If bacteria are spilled, turn off the Bunsen burner, soak up the majority of spill with a dry paper towel, discard towel as biohazard waste. Swab down the surface with 80%(v/v) ethanol and a fresh paper towel, then again dispose of paper towel as biohazard waste Section 5 – Repairs / Certification / Validation 1. Ensure rubber hoses on Bunsen burners are in good condition, and not frayed or corroded. Replace any damaged hoses. 2. Ensure fire extinguishers are regularly serviced Section 6 – Relevant safety data sheets and references 1. Read and understand SDS for ETHANOL, and Pathogen Safety Data sheet (or equivalent) for the microorganism used, if this is available. If no safety data sheet for the organism is available, consult the relevant scientific literature and/or ask your supervisor about the specific risks of handling the organism. 2. Risk assessment and SOP for “Use of Bunsen Burners” (SMB006), “Flammable liquids” (SMB013) and “Working with risk group 1 or 2 microorganisms” (SMB025 and SMB026).
SOP Consultation, Training and Approval Print names and enter signatures and dates to certify that the persons named in this section have been consulted/trained in relation to the development and implementation of this Standard Operating Procedure. WHS Representative (WHS Committee) certifies that consultation has taken place. Position Supervisor
Name
Signature
Date
employee / student employee / student employee / student employee / student Creation date: 25/10/2012 Last review date: 27/3/2015
Next review due: 27/3/2017
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employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student Name Authorising (Printed): DIANNE FISHER .......................................................
Signature: ..............................................................Date: 27/3/15 ............................ WHS Committee Representative Name (Printed): MARKUS HOFER ...................
Signature: ..............................................................Date: 27/3/15 ............................
Creation date: 25/10/2012 Last review date: 27/3/2015
Next review due: 27/3/2017
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