NP-40
PBS
Hazardous Substances Policy - Assessment
CHEMICAL HAZARD AND RISK ASSESSMENT
School of Biosciences
Name of supervisor / Dr.J.E.Turnbull / Assessment Number* / JETLW15#2055
Signature
Assessor / Laine Wallace / Date of Assessment / 10/07/02
Signature
Notes A School COSHH form in Word is available on the School Server.
Available from the Health and Safety Unit.
Guidance on making an assessment is given in Making a Chemical Hazard and Risk Assessment.
Guidance is also available from Guidance on Completing the Chemical Hazard and Risk Assessment Form.
Use a continuation sheet to expand any section of this form in hard copy version.
1 / LOCATION OF THE WORK ACTIVITY / 5th Floor Biosciences Tower2 / PERSONS WHO MAY BE AT RISK
List names where possible / Laine Wallace, Dr.J.E.Turnbull,K.Drummond,Z.Schofield,Dr.E.Yates, Dr,S Guimond, Dr.Z.Huang, Dr.A.Powell, J.Henstock, Dr. T. Kinnunen
3 / ACTIVITY ASSESSED / Wholemount immunostaining of worms with Bouin’s fixative
4 / MATERIALS INVOLVED / Attach copies of data sheet(s)
NAME / AMOUNT
max/stored / HAZARD / RISK PHRASES / HAZDAT NO*** / BIOSCIENCESNO***
NP-40 / 5ml / Harmful / R22 : Harmful if swallowed
R41 : Risk of serious damage to eyes
PBS / 500ml / Irritant / R36/37/38: Irritating to eyes, respiratory system and skin
Na-Borate buffer :
0.06M Borax
0.2M Sodium Hydroxide-
1M DTT / 500ml
33.3ml
5.5ml
20ml / Irritant
Corrosive
Harmful / R36/37/38: Irritating to eyes, respiratory system and skin
R34 : Causes burns
R21/22 : Harmful in contact with skin and if swallowed
R36/37/38: Irritating to eyes, respiratory system and skin
DAPI / 2ug / N/A / N/A
Antibodies / 200ul / N/A / N/A
Normal goat serum / 1ml / N/A / N/A
Bovine Serum Albumin
Mowiol / 2g
50ul / N/A
Irirtant / N/A
R36/37/38: Irritating to eyes, respiratory system and skin
R40 : Possible risk of irreversible effects
Bouin’s Fixative :
Picric acid
Formalin
Glacial Acetic acid / 2ml
75ml
25ml
5ml / Toxic / Highly Flammable
Toxic / Highly Flammable
Corrosive / Flammable / R2 : Risk of explosion by shock, friction, fire or other sources of ignition
R23/24/25:Toxic by inhalation, in contact with skin and if swallowed
R23/24/25:Toxic by inhalation, in contact with skin and if swallowed
R39/23/24/25 : Toxic: danger of very serious irreversible effects by inhalation, in contact with skin and if swallowed
R36/38 : Irritating to eyes and skin
R40 : Possible risk of irreversible effects
R43 : May cause sensitisation by skin contact
R35 : Causes severe burns
R10 : Flammable
5 / INTENDED USE**
Give brief details and attach protocol/instructions
NP-40 – constituent of wash buffer
PBS – constituent of wash buffer
Sodium borate buffer – to minimise fluorescent background.
Bouin’s fixative – to fix and preserve tissues for subsequent immunohistochemical investigation.
Mowiol – mountant for worms to adhere to slides and coverslips.
6 / RISKS to HEALTH and SAFETY from INTENDED USE
From personal exposure or hazardous reactions. Refer to OELs, flash points, etc., as appropriate. Are pregnant women, breast-feeding mothers especially at risk?
NP-40 – Risk of splashes to skins and eyes. Risk of serious damage to eyes.
PBS – risk of splashes to unprotected skin and eyes.
Sodium borate buffer –
Borax - Risk of inhalation, splashes to unprotected skin and eyes. . Risk of inhalation and skin contact with use of powder.
Sodium hydroxide – Risk of splashes to unprotected eyes and skin.
DTT – Risk of splashes of solution to unprotected eyes and skin . Risk of inhalation and skin contact with use of powder.
Bouin’s fixative –
Picric acid - Risk of inhalation, splashes to unprotected skin and eyes. Risk of fire. Risk of explosion by shock, friction, fire or other sources of ignition.
Formalin – Risk of inhalation, splashes to unprotected skin and eyes. Risk of sensitisation. Risk of fire. Risk of irrreversible effects.
Glacial acetic acid – Risk of splashes to eyes and skin. Risk of inhalation. Risk of fire.
Mowiol – Risk of contact with unprotected skin and eyes. Risk of irreversible effects.
7 / CONCLUSIONS ABOUT RISKS
Is level of risk acceptable? Can risk be prevented or reduced by change of substance/procedure? Are control measures necessary?
Due to the nature of some of the chemicals listed in this assessment there is a small risk . But due to the small amounts used and as long as measures laid out in section 8 are followed then the level of risk is acceptable. Non of the chemicals used can be replaced with other “ less hazardous” reagents.
8 / CONTROL MEASURES
Additional to Good Chemical Practice
Tremendous care should be taken when working with Picric acid not to allow it to dry out as it becomes incredibly explosive. Clean around bottle tops to avoid build up of crystalised picric acid. Avoid sources of ignition. Use in a fume hood.
Formalin should be used in a fume hood as it is toxic by inhalation. It is also a sensitiser. Use should be monitored. Avoid sources of ignition.
Glacial acetic acid should also be used in a fume hood as it is corrosive by inhalation and also flammable, so avoid sources of ignition.
Use of face mask for weighing out of powder chemicals to avoid inhalation of dust.
Always wear chemical resistant gloves, eye protection and a lab coat.
9 / INSTRUCTION/TRAINING
Specify course(s) and/or special arrangements.
One to one initial training.
10 / MONITORING
Performance of control measures,
Users of Formalin need to be registered with the HSU and Occupational Health to enable monitoring of exposure and possible subsequent sensitisation.
Instruction given as to importance and application of control measures. Training given to aid recognition of initial symptoms of sensitisation
Personal exposure / Health Surveillance
Users of Formailn needs to be registered with / As decided by Occupational Health representative
Occupational Health to monitor exposure.
11 / WASTE DISPOSAL PROCEDURE
See School Server for Approved Procedure Document on specific Chemical Waste Disposal.
Waste picric acid / Bouins fixative should be collected into a waste container, deposited with stores and disposed of by a specialist waste disposal company. DONOT ALLOW TO DRY OUT IN STORAGE!! – Inspect and add water every six months as needed. Rotate containers to distribute water every three months.
Formalin – Special waste as above
Other listed chemicals, at levels used within this protocol, can be disposed of down the sink with copious amounts of water.
12 / REVIEW
Enter the date or circumstances for review of assessment (maximum review interval 5 years)
10/07/02
13 / EMERGENCY ACTION
TO CONTROL HAZARDS / To stabilize situation eg spread absorbant on liquid spill; eliminate sources of ignition, etc.
Bouin’s / picric acid –Spill - Evacuate immediate area. Wear rubber boots and heavy rubber gloves. Contain spill. Use a spill response pad or pillow damp with water to absorb spilled material. Place pillows or pads in a non-metallic water impervious container with water added. Deposit absorbant pads with stores for specialised waste disposal. DO NOT LET DRY OUT!!!
Formalin – Evacuate immediate area. Wear rubber boots and heavy rubber gloves. Cover with drylime, sand or soda ash. Place in a covered container using non-sparking tools and transport outdoors ( outside Stores ) for specialised waste disposal. Small spills can be mopped up with tissue, tissues then left in a fume hood to evaporate before disposal in the general contaminated waste disposal procedure.
Acetic acid - Evacuate immediate area. Wear rubber boots and heavy rubber gloves. Cover with drylime, sand or soda ash. Place in a covered container using non-sparking tools and transport outdoors ( outside Stores ) for specialised waste disposal. Small spills can be mopped up with tissue, tissues then left in a fume hood to evaporate before disposal in the general contaminated waste disposal procedure.
DTT and Sodium hydroxide pellets and Borax powder - Evacuate immediate area. Wear rubber boots and heavy rubber gloves. Sweep up, avoid creating dust, hold in a bag, deposit with stores for specialised waste disposal. Large Liquid spill - Absorb on sand or vermiculite. Dispose of in the usual contaminated waster disposal procedure. Small spill - mop up with tissue and dispose of in the usual contaminated waster disposal procedure.
NP-40 – Large spill - Evacuate immediate area. Wear rubber boots and heavy rubber gloves. Absorb on sand or vermiculite and place in a closed container, deposit with stores for specialist waste disposal. Small spill - mop up with tissue and dispose of in the usual contaminated waster disposal procedure.
PBS – Large spill - Absorb on sand or vermiculite. Dispose of in the usual contaminated waster disposal procedure. Small spill - mop up with tissue and dispose of in the usual contaminated waster disposal procedure.
TO PROTECT PERSONNEL / Evacuation, protection for personnel involved in clean-up, Special First Aid
Ventilate area. Wear protective clothing where specified above.
First aid – Any chemical listed.
If swallowed, wash mouth out with plenty of water and call a doctor.
If inhaled – remove to fresh air – consult doctor if symptoms to do not alleviate.
In case of skin contact wash immediately with plenty of water, if symptoms persist then contact a doctor.
In case of contact with eyes, flush with copious amounts of water, for at least 15 minutes, assure adequate flushing by separating the eyelids with fingers. Call a doctor.
TO RENDER SITE OF EMERGENCY SAFE / Clean-up/decontamination
Cleanse area with a dilute decon solution and dry thoroughly.
CONTACT PHONE
10.10.00
* Prefix T is used for Teaching Assessment Number.
** Please include amount of chemicals used and how.
*** Hazdat No is the UNICOSHH datasheet report number.
Biosciences No is the Biosciences data sheet number.
UNICOSHH IS A CHEMICAL DATABASE ON THE HEALTH AND SAFETY UNIT SERVER.
BIOSCIENCES DATA SHEETS ARE AVAILABLE IN THE SCHOOL SAFETY OFFICE.
Whole mount immunostaining of worms in Bouin’s Fixative.
1. Wash the worms off the plate (10cm) with 3x15ml 0.5% NP-40/PBS, preferably ice cold.
2. Leave to settle by gravity for 15minutes.
3. Wash twice in 0.5% NP-40/PBS, spin on picofuge for 10secs in between, removing supernatant.
4. Ad 400ul Bouin’s Fixative, 400ul Methanol, 200ul 1M DTT.
5. Rock at RT for 30 minutes.
6. Snap freeze in LN2 and store at -70°C until use.
7. Thaw the worms under luke warm water ( freezing and thawing can be repeated once to enhance penetration of antibodies )
8. Rock for 30minutes at RT.
9. Spin on picofuge 10secs.
10. Wash three times with 0.5% NP-40, 200mM DTT in 20mM Na-borate buffer pH 9.6.
11. Spin in the picofuge for 10sec between washes, removing supernatant.
12. Add 1ml 0.5% NP-40, 200mM DTT in 20mM Na-borate buffer pH 9.6 and incubate for 2hrs.
13. Resuspend in 1% Goat IgG, 2% BSA, 0.5%NP-40 in PBS and incubate for 1hr.
14. Add Primary antibody into this mix of buffers and worms and incubate O/N at 4°C.
15. Spin 20secs in picofuge
15. Wash 4x15min with 0.5%NP-40/PBS
16. Apply secondary antibody on 1% Goat IgG, 2% BSA, 0.5%NP-40/PBS and incubate for I hour at RT.
17. Was 3x15mun with 0.5%NP-40/PBS
18. Was once with PBS. Include DAPI at 0.5ug/ml.
19. Spin in picofuge for 20 seconds, aspirate supernatant.
20. Add a small volume ( 15-20ul ) of Mowiol mount. Cut the end off a yellow tip and mount the worms on the slides using this. Usually cover with 18mm coverslips.
Bouin’s Fixative
75ml Picric Acid
25ml Formailin
5ml Glacial acetic acid
Store at +4°C. ( a bottle can be found in the cold room in Tarja’s box below JET work bench )
0.02M Na-borate Buffer.
33.3ml 0.06M Borax
5.5ml 0.2M NaOH
20ml 1M DTT
40.7 ddH2O.
After the addition of the DTT the final concentration will be 20mM Na-Borate
A 500ug/ml DAPI Stock solution in 1.5ml eppendorfs wrapped in foil is at +4°C in Tarja’s shelf in a grey box in the fridge.
Aliquots of Mowiol in blue 0.5ml tubes are at -70°C in Tarja’s white square box with clear lid. ( The box with no writing on it ) I prefer Mowiol to any commercial mounting agent. Once the aliquot is thawed it can be used for a day or two, no longer.