SAFE WORK PROCEDURE
Activity/Task: Preparation of Microbiological growth media
SWP No: SWPOR0032
Trim No:
Version No:
Date Signed Off:
Approved: Yes / No
RISK IDENTIFICATION AND CONTROLS:
Please include all steps involved in the performance of the task
NOTE: All PPE required must be listed and the minimum PPE for each chemical must be listed as per the relevant SDS.
PROCEDURE STEPS
1.
Scope:
This SWP outlines the correct procedure and preparation of microbiological growth media in Faculty of Science Laboratories
2.
Objectives:
To instruct staff and students in the safe preparation of microbiological growth media
3.
References:
Safety in the Laboratory 2243.3
CSU Biosafety Manual
SWP Magnetic stirrer and hotplate
Safe Work Procedure
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SWP for intended autoclave
SWP for use of Biosafety cabinet/laminar flow cabinet
4.
Timing:
Whenever media is being prepared
5.
Responsibilities:
Technical Staff:
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Staff must be trained and competent in the technique prior to unsupervised use (sign this SWP)
Technical staff may have to train research students or academic staff in the procedure
Ensure this procedure is reviewed regularly and updated when changes are required or there is an incident
Ensure PPE is available and used
Managers


Manager is required to sign off on training competency of technical staff
Ensure plant and equipment is purchased and well maintained
6. In the event of: (emergency or other) First Aid officer available on campus or 000 where necessary. First Aid kit available in the Laboratory and Biological Spill Kit
stocked and ready in the Laboratory
6.
Location:
Science Laboratories
8. Training Requirements:
Persons shall not prepare media until they are deemed competent by the Laboratory Manager and have read and signed this SWP.
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9. Plant and Equipment Details:
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Schott bottles or flasks
Magnetic stirrer and stir bar
Water bath
Powder funnel
Autoclave taped
Biosafety or laminar cabinet (optional)
Autoclave
Recipes
Sterile petri dishes / slopes jars
Dehydrated Growth media
10. Engineering Details, Certificates, Work Cover Approvals:
Biosafety cabinet requires annual certification
Autoclave required Workcover certificate
Autoclave required annual testing and certification
11. Maintenance Requirements:
Annual electrical Test and tag of electrical items
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12. Task/Activity Steps:
1:
Ensure work area is clean and obtain all PPE and equipment necessary. Read through SDS’s and SWP as required
Possible Hazards:
Safety Controls:
Risk Score:
Fine powders
Clean laboratory coat/gown
Safety glasses/ face shield
Dust mask
Gloves
Remove ignition sources and have fire blanket and extinguisher in
Lab
5
Chemical risks associated with media, see SDS’s for each media powder in use
Bunsen burner
70% Ethanol
2: Before you start
Calculate volumes required based on
Agar plates = 45 / litre
Slopes = 12-15mls each
Deeps = 18mls / tube
Broths as stated in individual lab protocol/recipes
Prepare media in manageable batch sizes. Agar for pouring plates should be in batches that are not too heavy for the individual to pour from (suggest no more that 500ml
bottles). Media can be made in larger amounts if space in the autoclave is an issue, but needs to be aliquoted into sterile 500 ml bottles prior to pouring plates.
Possible Hazards:
Safety Controls:
Risk Score:
Using quantities too heavy to hold for extended period, particularly when
hot
Prepare in batches of manageable quantities dependant on the
strength of the individual
6
3: Prepare the batch of media by;

Add spin bar and powder funnel to appropriately sized Schott bottle or flask.
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Weigh out required amounts of ingredients into and transfer into bottle or flask. Wear gloves mask and eye protection. Follow recipes closely and tick ingredients off as
they are added.
Add a small amount of RO / de-ionised water to the container. Put on lid and swirl to mix, until no dry powder is discernable. Add RO / de-ionised water to final volume
required and loosely re-apply lid.
If preparing plates or any other media to be sterilised and aseptically dispensed, apply autoclave tape to lid and label with media type. Autoclave according to media
recipe and autoclave SOP.
Remember to autoclave any equipment or glassware required for aseptic dispensing at the same time as the media.
ALWAYS wear thermal gloves when removing media bottles
Risk Score:
Possible Hazards:
Safety Controls:
Steam /heat burns
Fine media particles
4:
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Thermal Gloves are required for handling media bottles from the
Autoclave
Face mask required when handling dry media
5
Slopes or Agar Deeps
Weigh out reagents and add water as above.
Dissolve agar completely by boiling (do not leave unattended) or by autoclaving at 105ºC for 5 to 10 minutes.
Keep warm and well mixed. Dispense into final containers, cap loosely, then sterilise (autoclave) according to media recipe.
For slopes.
After sterilising and when cool enough to handle, but not yet set, lay on the bench with lid resting on a block that is the correct height to achieve the desired slope on the
media. (General purpose slopes, use red test tube rack)
Possible Hazards:
Safety Controls:
Risk Score:
Steam /heat burns
Thermal Gloves are required for handling media bottles from the
Autoclave
Face mask required when handling dry media
5
Fine media particles
5: Broths

When the ingredients have dissolved dispense into required containers, cap and autoclave as for Slopes and deeps.
Possible Hazards:
Safety Controls:
Risk Score:
Thermal Gloves are required for handling media bottles from the
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Steam /heat burns
Autoclave
Face mask required when handling dry media
5
Fine media particles
6: Pouring Agar Plates
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After media is started off in the autoclave, prepare the biosafety/laminar cabinet according to the SWP if using, or for routine pouring, swab bench with 70% alcohol. Turn
on water bath with temperature set at 50-55°C.
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When media is sterilised and whilst still hot place it in the water bath.
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With freshly washed hands, put on gloves and spray with 70% alcohol. Prepare Petri dishes by removing from the plastic bag and stacking ready for use in laminar flow
cabinet or on right hand side of bench, lid uppermost, 10 plates per stack.
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Retain plastic bags for storing poured plates.
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Maintain sterility at all times and aseptic technique.
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When media has cooled to a manageable temperature, remove a container of media from the water bath and spray the outside of the container with 70% alcohol, place
on stirrer element and stir slowly to avoid creating air bubbles. NB if media is too hot when poured, this will lead to excessive condensation.

Place in laminar flow cabinet or on bench close to Bunsen burner.
When pouring plates on the bench:
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Work close to Bunsen and reasonably quickly to avoid media in bottle setting.
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Start pouring plates by placing a stack of plates from the right of the bench (opposite if left handed), in front of Bunsen burner.
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Remove the lid from the media and flame neck of bottle. Take care to always hold bottle at a 45º angle to reduce risk of contamination and periodically flame neck and
opening. Remove the lid from a Petri dish and hold above, shielding media from contamination. Pour in approx. 20 – 25mls of media (or as required in recipe), replace lid
and then place the filled dish on top of previously poured plate, creating stacks of 10 plates. Carefully push the completed stack to the back left hand side of the bench.
Air bubbles should be removed touching with a hot, sterile loop or wire or by flaming with a Bunsen burner. Leave plates to cool and set. Stacking plates allows the plates
to cool more slowly and reduces condensation.

Leave on bench overnight to dry, if very wet may be left longer.
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Label the bottom of the dish, invert (so agar is uppermost and lid is on bottom) and place back in plastic bag in stacks of 20 and label bundles with media type and date
prepared.
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Store at 4ºC, inverted (so agar is uppermost and lid is on bottom) in media fridge. Do not store in domestic refrigerator, as this causes excessive condensation.

Blood agar plates should ideally be stored for no longer than 4 weeks, other plates will depend upon additives (see method). Simple media can be stored for longer
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periods, but length of storage may affect quality and lead to excess condensation.
When pouring plates in the cabinet:
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Work reasonably quickly to avoid media in bottle setting.
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Start pouring plates by working from the left of the cabinet (opposite if left handed).
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Remove the lid from the media. Remove the lid from a Petri dish and hold above, shielding media from contamination. Pour in approx. 20 – 25mls of media (or as
required in recipe), replace lid and then place the filled dish on top of previously poured plate, creating stacks of 10 plates. Then push the filled stack to the back of the
cabinet. Air bubbles should be removed by sucking them out with a sterile transfer pipette. Leave plates to cool and set.
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Take from cabinet, label the bottom of the dish, invert (so agar is uppermost and lid is on bottom) and place back in plastic bag in stacks of 20 and label bundles with
media type and date prepared.
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Store as above.
Possible Hazards:
Safety Controls:
Risk Score
Ignition with Ethanol and the Bunsen burner flame
Ensure the ethanol container is placed away from the Bunsen burner
or back in the flammables cupboard before the Bunsen burner is
used. Give enough time for fumes of ethanol to dispenses prior to
igniting a match /lighter
5:
7: When you finish

Soak empty containers with hot water to loosen set agar before washing up.
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When cabinet has been emptied wipe down with 70% alcohol as per Laminar flow/ Biosafety cabinet SWP.
Possible Hazards:
Safety Controls:
Risk Score:
6
8: Drying plates
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If agar surface of plates needs to be especially dry (eg for drop or spread plates), plates can be dried by the following methods;
For large class size quantities of plates, take plates out of plastic bag, place in plate rack (agar uppermost and lid on bottom) and place in 37ºC incubator overnight.
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For small quantities of plates, place open plates in 37ºC incubator for 1 hour or until dry. Plates should be placed so agar surface and lid are both facing downwards in
incubator. To save space, lid may be rested over edge of agar plate at 45º angle.
If plates required to be dry, it is best to pour, leave on bench for several days, dry and use immediately, rather than refrigerate and store.
Safe Work Procedure
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SAFE WORK PROCEDURE:
Assessment dates
Initial assessment date: adapted from SOPOR032
Current assessment date: 6/11/13
Reassessment due date: 6/11/18
Assessors:
Name: Ingrid Stuart
Signature:
Date: 6/11/13
Grace Kay
6/11/13
Recommendation: (Technical Officer/Supervisor/Manager)
Follow up required: Yes / No
Name: Leonie Diment
Date: 6/11/13
Signature:
Approval: (Facility Manager, Head of School, Manager University Laboratories)
Name: Kellie Munn
Safe Work Procedure
Version 1: Sept 2012
Signature:
Date: 7/11/13
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Risk Assessment Matrix:
Evaluate the level of risk associated with the hazard identified.
SEVERITY
LIKELIHOOD
How severely could it hurt someone
How likely is it to be that bad?
or
How ill could it make someone?
!!!! Kill or cause permanent
disability or ill health
++
+
-
--
Very likely
Could happen at any time
Likely
Could happen sometime
Unlikely
Could happen, but very
rarely
Very unlikely
Could happen, but probably
never will
1
1
2
3
!!!
Long term illness or serious injury
1
2
3
4
!!
Medical attention and several days
off work
2
3
4
5
!
First aid needed
3
4
5
6
Safe Work Procedure
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I have read and understand this Safe Work Procedure:
Name:
Safe Work Procedure
Version 1: Sept 2012
Position/Role:
Signature:
Date:
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