<p> Glycerol 10% Glycerol Bacterial strain (E. coli)</p><p>Bacterial culture grown overnightHazardous Substances Policy - Assessment CHEMICAL HAZARD AND RISK ASSESSMENT School of Biosciences </p><p>Name of Steve Busby Assessment Number* SJWBJM#2092 supervisor Signature Assessor Jennie Mitchell Date of Assessment 08/12/02 Signature</p><p>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.</p><p>1 LOCATION OF THE WORK ACTIVITY G3/T102</p><p>2 PERSONS WHO MAY BE AT RISK List names where possible All lab members (see current lab members list at front of folder).</p><p>3 ACTIVITY ASSESSED Preparing electrocompetent bacterial cells and their use in electroporations.</p><p>4 MATERIALS INVOLVED Attach copies of data sheet(s) NAME AMOUNT HAZARD RISK PHRASES HAZDAT NO*** BIOSCIENCESNO*** max/stored Glycerol 2.5 litres IRRITANT R36 Irritating to the eyes 354 10% Glycerol 800ml N/A Bacterial strain (E. coli) 1colony See GMMO assessment. Bacterial culture grown 5ml See GMMO overnight assessment. Exponential phase 400ml See GMMO bacterial culture assessment Plasmid DNA mg-g qnty See GMMO assessment SOC media i.e 200ml N/A 2% Bactotryptone 0.5% Yeast extract 10mM NaCl 2.5mM KCl 10mM MgCl2 10mM MgSO4 20mM Glucose Antibiotics Please see preparation of antibiotics for details of risks # Ampicillin solution 5ml HARMFUL R42/43 May cause 100mg/ml sensitisation by inhalation and skin contact. Tetracycline solution 5ml FLAMMABLE R11 Highly flammable 25mg/ml in 50% ethanol TOXIC R22 Harmful if swallowed R40 Possible risk of irreversible effects R63 Possible risk to the unborn child R64 May cause harm to breastfed babies R36/37/38 Irritating to eyes, respiratory system and skin. R33 Danger of cumulative effects. Kanamycin solution 5ml TOXIC R61 May cause harm to 30mg/ml the unborn child. LB amp plates i.e 1-2 litres N/A None but avoid making 1% Bactotryptone worth are dusts which may irritate 0.5% yeast extract stored the mucous membranes 0.5% NaCl and the respiratory system 1.2% Agar 100ug/ml Ampicillin or other antibiotic</p><p>5 INTENDED USE** Give brief details and attach protocol/instructions A culture of E.coli is grown up and then spun down resuspending the pellet in a gradually smaller volume of 10% glycerol. See attached sheet. A small amount of cells are then placed in a special electroporator cuvette along with the plasmid DNA of interest and a large voltage is passed through them allowing the cells to take up the DNA. After incubating for a short while the cells are plated onto selective media. </p><p>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? There may be a risk of irritation of the mucous membranes and respiratory system from the fine powder used to make SOC and the LB plates. There may be an electrocution risk from the electroporator machine. There is a risk of irritation of the eyes from glycerol. Risk of skin contact and possible inhalation of all the powders (Ampicillin, Tetracycline, and Kanamycin). Risk of skin contact or spashes to the eyes with the solutions (Ampicillin, Tetracycline, and Kanamycin).</p><p>7 CONCLUSIONS ABOUT RISKS Is level of risk acceptable? Can risk be prevented or reduced by change of substance/procedure? Are control measures necessary? Good Chemical Practice should cover the risks from most subtances, although rough handling when weighing out the LB agar components could produce a health risk. When unloading the autoclave there may a risk of pressurised containers exploding or forcing the tops off. The electrocution risk should be considered. Pregnant or breastfeeding women should avoid contact with Tetracycline, Chloamphenicol and Kanamycin. GCP may not be enough to protect other people and so control measures should be used for all antibiotics.</p><p>8 CONTROL MEASURES Additional to Good Chemical Practice When weighing out the media care should be taken so that a dust is not generated. Gentle handling should avoid this. Make sure the tops of bottles of media are loose when they go into the autoclave and do not open until the pressure has reached zero. Check the connections on the electroporator before beginning and make sure that it is electrically tested regularly. Avoid touching the eyes when handling glycerol. Gloves should be worn at all times when handling antibiotics. Pregnant women may prefer not to handle these antibiotics.</p><p>9 INSTRUCTION/TRAINING Specify course(s) and/or special arrangements. N/A</p><p>10 MONITORING Performance of control measures, Users of Ampicillin andTetracycline need to be registered with the HSU and Occupational Health to enable monitoring of exposure and possible subsequent sensitisation. Personal exposure Users of Ampicillin, Tetracycline and Health Surveillance As decided by OH represtative. need to be registered with Occupational Health to monitor exposure.</p><p>11 WASTE DISPOSAL PROCEDURE See School Server for Approved Procedure Document on specific Chemical Waste Disposal. The supernatants produced after spinning the cells down should be autoclaved or treated with VIRCON prior to disposal. Waste agar plates that had E. coli grown on them have to be autoclaved before they can be disposed of. Other waste antibiotic solutions can be disposed of down the sink with plenty of water as the amounts are likely to be very small, <1ml.</p><p>12 REVIEW Enter the date or circumstances for review of assessment (maximum review interval 5 years) 08/12/07</p><p>13 EMERGENCY ACTION TO CONTROL HAZARDS To stabilize situation eg spread absorbant on liquid spill; eliminate sources of ignition, etc. Spills of glycerol should be mopped up with paper towels. Spills of bacterial cultures should be treated by covering with VIRCON. Small spills can be treated by applying 70% ethanol to the area although care must be taken to shut off all sources of ignition. In the event of accidental release of Tetracycline, Ampicillin or Kanamycin self-contained breathing apparatus, rubber boots, heavy rubber gloves should be worn. Disposable coveralls should be worn and discarded after use. The material should be swept up and then placed in a sealed container and treated as special waste for disposal. Any spill kit used must be treated as special waste for disposal. TO PROTECT PERSONNEL Evacuation, protection for personnel involved in clean-up, Special First Aid Protective clothing and gloves should be worn during the clean up procedure. Ventilate areas at all times. First aid – Any chemical listed If inhaled - remove to fresh air – consult doctor if required. In case of contact with skin, wash immediately with plenty of water – consult doctor if required. In case of contact with eyes, flush with water for 15 mins ensuring adequate flushing by opening of eyelids with fingers.</p><p>TO RENDER SITE OF EMERGENCY SAFE Clean-up/decontamination After the site of a bacterial spillage has been disinfected the area can be mopped up and washed with water. Any other spill sites can also be washed with water.</p><p>CONTACT Prof Steve Busby PHONE 414 5469</p><p>10.10.00 * Prefix T is used for Teaching Assessment Number.</p><p>** Please include amount of chemicals used and how.</p><p>*** Hazdat No is the UNICOSHH datasheet report number.</p><p>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.</p>