Published on January 19, 2008
LABORATORY PRACTICEAims of this course:: LABORATORY PRACTICE Aims of this course: To aid understanding of basic good laboratory practice To discuss practical tips for maximising research effort To explain the fundamentals of cellular and molecular biology techniques Session (1): Session (1) Good laboratory practice - what is it and why should we bother? Requirements for safe working practice – including COSHH, risk assessment, biohazard containment, genetic manipulation Practical tips for maximising research effort Session (2) - Fundamentals of key cellular and molecular biology techniques: Session (2) - Fundamentals of key cellular and molecular biology techniques Cell biology Tissue culture Flow cytometry Molecular biology PCR protein analysis Good laboratory practice: Good laboratory practice Strict Definition: “A quality system concerned with the organisational process and the conditions under which non-clinical health and environmental safety studies are planned, performed, monitored, recorded, archived and reported.” (Organisation of Economic Cooperation and Development) How does GLP relate to academic research?: How does GLP relate to academic research? Safety Reproducibility Reporting 1) Safety – your responsibilities: 1) Safety – your responsibilities Those who work in the University should be aware of the University’s Statement of Safety Policy (http://www.nottingham.ac.uk/safety) Everyone must know the risks and potential hazards of the materials and equipment he is working with and so be alert to any dangers. You should also:: You should also: Have had a safety induction & training – check the local rules in all laboratories that you work in Know who your Safety Officer is – seek advice, report any incidents Consider your health status (e.g. allergies) & any vaccination requirements (e.g. Hepatitis B) Complete risk assessments so that you know how to use, store and dispose of all chemicals how to deal with any spills or accidents the safety precautions required and for some equipment, e.g. safety cabinets, how they work Risk Assessments: Risk Assessments Hazardous Chemicals - Control of Substances Hazardous to Health Regulations 2002 (COSHH). Biological Hazards – Genetic manipulation, handling pathogens, Crime & Security Act 2001 Radiochemicals – Contact the safety office for permission before use. Experimental risk assessments – Combination of hazards associated with a technique or protocol. COSHH assessment: COSHH assessment Access your School/Department database Complete the assessment based upon MSDS Identify the hazard, conditions for storage, use, disposal, emergency procedures This should be completed before ordering the chemical Amounts and concentrations make a big difference. Material safety data sheets: Material safety data sheets Download information from the supplier’s website or Go to your school’s COSHH database or Look at other websites, e.g. http://physchem.ox.ac.uk/MSDS/#MSDS http://hazard.com/msds2/ Example COSHH assessment: 1M HCl: Example COSHH assessment: 1M HCl √ √ √ √ √ √ √ Hazard symbols: Hazard symbols You must be able to recognise and explain the significance of the following hazard symbols. Look for them around the laboratory – all your chemicals should be labelled appropriately Oxidizer Harmful Biohazard Corrosive Toxic Radioactive Highly flammable Personal protective equipment: Personal protective equipment But also be aware of hazards from your work possibly affecting others working around you – use a fume cupboard, warn others, restrict access to where you are working. Safety cabinets: Safety cabinets Fume cupboards Designed to exhaust fumes from the laboratory environment. Class I biosafety Protects the user from aerosol hazards Slide15: Class II biosafety Protects both the user and the material within the cabinet Class III biosafety User works through gloves. The material within the cabinet is protected by hepa-filtered air.The inside of the cabinet operates at a negative pressure. Biological hazard Categories: Biological hazard Categories HAZARD GROUP 1- Agent is unlikely to cause human disease HAZARD GROUP 2- Agent can cause human disease and may be a hazard; unlikely to spread to the community; effective prophylaxis or effective treatment available. HAZARD GROUP 3- Agent can cause severe human disease and presents a serious hazard; may present a risk of spreading to the community; effective prophylaxis or treatment available. HAZARD GROUP 4- Agent causes severe human disease and is a serious hazard; likely to spread to the community; no effective prophylaxis or treatment available. (None at the Univ. of Nottingham.) Refer to ACDP handbook for categorization & regulations. All work with Group 3 pathogens must be with permission from the safety office Biohazardous waste must be rendered safe before disposal: Biohazardous waste must be rendered safe before disposal Clinical waste Sharps Disinfection Autoclave Blood and human tissue: Blood and human tissue Particular concern over the potential presence of infectious agents including Hepatitis B & HIV. It is recommended that any worker be successfully immunised against HepB before commencing work with human/primate samples. Usual recommendations: Check your risk assessment & local rules before starting, Assess the HepB/HIV risk in samples, Work under biocontainment conditions (safety cabinet), Take care in disposal (e.g. double-bag & autoclave prior to disposal with clinical waste) Genetically modified organisms: Genetically modified organisms Genetic Modification is defined as the altering of the genetic material of an organism by a way that does not occur naturally by mating and/or natural recombination. (Genetically Modified Organisms (Contained Use) Regulations 2000) It is the policy of the University that all work involving GMOs is carried out in such a way as to prevent undue risk either to those carrying out the work, to those who may otherwise be affected by the work, or to the environment. Genetically modified organisms: Risk assessments for new projects must be completed and submitted to a GM Safety Committee hazard identification • estimation of the severity of the consequence • determination of a provisional containment class • environmental considerations • estimation of likelihood of harm occurring • can all hazards be properly controlled Results in assignment of the final containment and activity classification 1-3 (classes 2 & 3 notifiable to HSE) Genetically modified organisms Genetically modified organisms: Work must be carried out in a suitably equipped and designated containment laboratory Periodic reviews of risk assessments done Health surveillance may be required Records of exposure may be required Emergency plans are needed Genetically modified organisms Other hazards in the laboratory:: Other hazards in the laboratory: Allergens Ionising radiation Sound Electricity Sparks & naked flames Configuration of computer workstations Lifting General lab safety rules: General lab safety rules Fastened laboratory coats must be worn in the laboratory Appropriate footwear must be worn (no open-toed sandals) Smoking, eating, drinking and application of cosmetics is not permitted Cuts and abrasions must be covered with a water proof dressing. Long hair should be tied back. COSHH risk assessment forms must be completed for all procedures. Gloves and eye protection should be worn when dealing with hazardous substances. Staff must have knowledge of the necessary emergency procedures Mouth pipetting is forbidden. Solvents should be stored in clearly labelled spark-proof cupboards. Corrosives should be stored in clearly labelled corrosives bins. Toxins should be stored in a locked poisons cupboard. When using powders, avoid all contact and avoid raising dust. Dispose of all sharps in a sharp-safe box. Never attempt to use any equipment unless you have undergone a full course of instruction 2) Record keeping: 2) Record keeping Record keeping in GLP: Record keeping in GLP Detailed records will help in your data analysis, repeat experiments, optimisation, and thesis preparation. Recorded documentation of what was done & when - proof that cheating nor falsification of results occurs. All processing of personal data must comply with the terms of the Data Protection Act 1998. Data must be retained intact for a period of at least seven years from the date of any publication which is based upon them. May need special arrangements e.g. for commercially funded research. Intellectual property. 3) Reliability of Data: 3) Reliability of Data Considerations for good experimental design:: Considerations for good experimental design: Controls needed Positive Negative Internal standards Statistical analysis of your results Number of replicates Paired or unpaired data Experimental errors: Experimental errors Systematic errors – e.g. instrument errors. These do not contribute to the spread of data. Random errors – e.g. fluctuations in temperature. These do affect the spread of data. Careless errors – e.g. pipetting errors. These also affect the spread of data. Accuracy - how close a measurement is to the accepted value Precision - how reproducible your results are Consider:: Consider: Why may experiments fail or give inconclusive results? What could cause inconsistencies between experiments? What could cause inconsistencies within an experiment? What measures could provide more accuracy? What measures could provide more precision?