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Team:UCL/Project/Safety
From 2013.igem.org
KEEPING SAFE
It's Just Good Lab Practice
The health and safety considerations of the project are vital for the well-being of the project members and the laboratory environment. Some of the key considerations to be made involve handling bio-hazardous materials, process chemicals and mechanical operations; requiring planning and awareness in order prevent hazards from occurring, as well as appropriate plans of action in order to deal with any situation that may occur.
The Escherichia coli strain used is not considered pathogenic, and thus not of considerable risk to the environment or team members. Despite this, there must still be a level of Good Laboratory Practice (GLP) to reduce risk associated with performing experiments on a daily basis. As a minimum requirement, all members will wear some form of protective clothing, which generally consists of a lab coat and goggles, as well as single use gloves.
For disposal of spent materials, any item should be isolated and sealed in a container which prevents physical contact with any of the facility. The material should then be removed from the room through the waste corridor and disposed of accordingly (chemical or steam treatment for example).
Continual monitoring and safety reviews of the facility is required to maintain GLP and the condition of the plant. By undertaking continual assessments of laboratory safety, the risk of a hazard occurring in the plant can be reduced. Training of team members in safe operation in general safety protocols is essential to reduce risk; so safety training and education in good laboratory practise was undertaken by all team members before experiments could begin.
The main principle behind performing experiments in a safe environment is to minimise risk. Risk is considered as the amalgamation of the potential damage that a hazard could cause, combined with the likelihood of said hazard occurring. The damage a hazard could cause is normally set by the equipment present in the laboratory, whilst laboratory team members can often decrease risk by decreasing the likelihood of a hazard occurring. Below the three main categories (Microbiological, Chemical and Mechanical) are defined with tables showing which hazard fall into said category, and how the risk is minimised in each case.
Microbiological Hazards
The key focus of microbiological hazard control concerns the Escherichia coli expression system being used. The majority of Escherichia coli strains are categorised into Bio-safety levels 1 or 2, with non-pathogenic strains placed into level 1 under World Health Organisation classifications. Therefore the Escherichia coli strain used in the facility is considered to be Bio-safety level one, which often requires less stringent safety standards. The genetically engineered strains also may require more safety regulation requirements than wild type micro-organisms; however advances in synthetic biology are allowing more control over the expression system in terms of pathogenicity and also the ability to express suicide genes, which can prevent any live Escherichia coli from surviving outside of the facility environment. Regardless, the facility must ensure and validate that all of the Escherichia coli is killed and effectively disposed of. Pressure systems and directed ventilation are in place to prevent escape of any strains, although this may lead to possible contaminants entering the halls, which dictates that the live Escherichia coli must be sealed in containers or vessels at all times where possible in order to prevent infection. The use of mammalian systems in the project also present a degree of hazard, although the associated risk is lower when compared to using bacteria.
| Hazard Type | Hazard Explanation | How Hazard Is Addressed |
|---|---|---|
| Escape of Escherichia coli from lab | The strain(s) exit the laboratory environment, thus allowing the possibility of contamination of exterior objects and/or persons. | Pressure of lab is lower than of corridor, thus causing airflow into the lab as opposed of out, which minimises the chance of airborne or aerosol escape of E. coli. Laboratory coats are worn over clothes which do not leave the lab, thus reducing possibility of a team member inadvertently acting as a carrier out of the lab. |
| Contamination of E coli/ HeLa/ Microglia | Contamination of the desired strain leads to competition between several strains of bacteria, resulting in inaccurate stocks and therefore unusable data. Note that bacterial and mammalian experiments are undertaken in separate parts of the facility. | Exposure of strain to open air is minimised, which is standard practise for any container that may be involved with the desired E. coli strain. Single use gloves are used to minimise possible contamination when experiments are performed. Ethanol is applied on work surfaces before and after experimental procedures in order to minimise contamination on work surfaces etc. |
| Contamination of team members | Illness could ensue from working in the laboratory environment. | Team members are not advised to work when feeling unwell. Gloves, lab coat and goggles are also worn to minimise contact with organisms. Personal hygiene standards are advised to be upheld in particular for laboratory workers. |
| HeLa Cells | Level 2 biosafety class cells | Cell experiments always performed in a sterile environment and a fume hood, with a supervisor providing assistance to the experiment when necessary |
| Microglia cells | Level 2 biosafety class cells | Dr. Darren Nesbeth will perform the neccesary assays in place of iGEm members due to the safety regulations |
Chemical Hazards
When performing experiments, it may be necessary to use dangerous chemicals during certain processes. Whilst using hazardous chemicals is avoided where necessary, in some cases it is required, so in each case where this occurs the procedure must be executed in a fashion which is as safe as reasonably possible. Therefore standard protocols such as wearing protective clothing may be added to by performing experiments in fume hoods or other such devices to minimise any chance of contact via evaporation etc.
| Hazard Type | Hazard Explanation | How Hazard Is Addressed |
|---|---|---|
| Ethidium Bromide (EtBr) | Mutagenic. Minor toxicity issue | All operations using EtBr are carried out in a fume cupboard - separate equipment (pipettes etc.) are used only in the fume cupboard specifically for EtBr. Gloves and long sleeved protective gowns MUST be worn, disposal of items in contact with EtBr are disposed of separately to other wastes. Wash hands after gloves are removed. |
| High concentration Ethanol (EtOH) and other alcohols | Flammable. | Securely stored (glass container) in a separate cupboard in volumes less than 2 Litres. |
| Dimethyl sulfoxide & Zeocin | Irritant to operator if contact with skin is made | Chemical handled with extreme caution, with particular attention to no skin exposed when performing an experiment, particularly goggles must be worn |
Mechanical Hazards
For this project, mechanical hazards pose the lowest probability of occurring out of the three and thus may be considered as the lowest risk group. However, there are hazards present which must be minimised. The predominant issue here is the use of the desktop centrifuge system, which poses considerable danger if a blowout occurs.
| Hazard Type | Hazard Explanation | How Hazard Is Addressed |
|---|---|---|
| Centrifuge | Centrifuge mechanical failure (eg. of rotor) can cause severe damage to the machine, and possibly personnel in the nearby vicinity. | Regular inspections and maintenance of the machine is required (performed by external engineers). Machine not to be used if in non optimal condition. Centrifuge must be balanced to minimise chance of rotor failure. Centrifuge must be properly closed before use (required for the machine to work.) Attention must be paid to ensure that nothing is spilled in the centrifuge bowl, or a build up of dust or aerosol should also be avoided. |
Concluding Comments
The safety of the team members, produced material and environment are key aspects to the smooth running of the project, and it must be ensured that adequate measures are in place to provide protection and ensuring that guidelines can be followed proceeding mandatory safety training. Current Good Laboratory Practice must also be adhered to at all times. By following guidelines and acting in an appropriate manner whilst in the laboratory, the general risk in terms of micro-biological, chemical and mechanical aspects will be reduced to a level which is as low as reasonably possible for all project members involved.
Due to the relatively small scale of the experiments performed, the quantities used of material is not large, but still must be handled and respected properly at all times. Proper containment and labelling of materials in sealed containers is necessary, especially when of particular hazard such as live material or Ethidium Bromide. Flammability concerns are also present with alcohol and other flammable materials stored in the lab, although the volumes (generally less than 2L) are not considerably large enough, although should still be used and handled properly when used, with fire extinguishers available and also emergency exits present if required. Protective clothing also should be worn where appropriate to minimise the possibility of skin contact, with wash and first aid station available in key areas where allowable due to the area classification.
The mechanical safety predominantly concerns the proper maintenance of the centrifuge system, which can cause a considerable risk if a failure occurs. Regular inspections should be made, with the centrifuge often restricted access when it was considered unfit for use. This room is also placed away from mammalian and bacterial processing centres. Prevention methods should also be in place such as emergency shut-off procedures.
By following all guidelines set out in safety training and following supervisor instructions, the project may be performed in a safe and effective manner. Continual training and inspection of the facility will aid in ensuring safety levels are maintained, with review protocols in place to investigate any faults. Team members have been trained in general safety, with supervisors having first aid experience and training. These considerations combined will ensure the safety risk to the personnel; product and environment is as low as reasonably possible. For further reading, the Institute of Chemical Engineering (IChemE) has relevant information, as well as other safety guidance documents relating to the relevant country where the university is located.
Safety Forms
