LBL02001

Laboratory Induction

Author: S. Thompson	Approved by:	SOP No. LBL02001
Signed:	Signed:	Effective from:
Date:	Date:	Last edited:

• • TODO**: SOP for "storage and recovery of frozen bacterial stocks and procedures" required -- ref from section 6.15

• • TODO** SOP for gel visualisation (search 'gel vis')

• • TODO**: List of MSDSes (search page)

• • TODO**: List of chemicals (search page)

• • TODO**: PCR docs link for 6.5 PCR Machine

1. Purpose

This describes the laboratory induction procedure for all new users of the laboratory facility. It is a requirement that this be completed by all new laboratory users and their confirmation of its requirements recorded by signing the appropriate training sheet.

2. Scope

This applies to and is required by all users of the laboratory. If there are any questions please contact the biological safety officer (BSO).

3. Responsibilities

Users of the laboratory, by being inducted by the biological safety officer, are hereby acquiring the responsibilities of individuals working in a laboratory environment. They should be made aware of and agree to be mindful of the hazards and risks that are or could potentially be present within a biological laboratory working to containment level one standards. Further, for all procedures, the laboratory user should be aware of all other SOPs that address the kind of procedures they wish to carry out. The principal investigator for each project is required to be aware of any risks involved in carrying out their proposed project before work begins. In the case of a proposed project involving work with genetically modified organisms (GMOs), a written risk assessment must be carried out and approved by a biosafety expert designated by the BSO. In case of any doubt please ask the BSO for further information.

The current BSO is Samantha Thompson. Contact: bioscisam@gmail.com , tel 07720677678.

4. Related documents

All SOPs relevant to further work to be carried out.

TODO SOPs don't need to be read/understood at the time of induction IIUC, so their relevance here is mostly around them being required reading for the inductee prior to commencing any work. Yes? this and 6.1 need minor changes to reflect that if so.

5. Definitions

BSO – Biological Safety Officer
GM – Genetically Modified
GMO – Genetically Modified Organism
GMSC – Genetic Modification Safety Committee

6. Procedures

6.1 Laboratory Access Control

The lab can be accessed using the number lock or using the card sensor to unlock the door, if your access card (such as an oyster card) has been registered. Access control to the laboratory area is to ensure that only trained and responsible individuals can make use of the laboratory facilities. Those wishing to access the laboratory should only do so after going through and understanding the safety and operational factors highlighted in this document. This induction process should be conducted by the Biological Safety Officer (BSO). Any relevant LBL Standard Operating Procedures (SOPs) should be read and understood where this pertains to the work the user desires to carry out. Lab users should be conscious of the safety requirements and skill level necessary when working with reagents and equipment needed in their particular projects and should consult the SOP documentation and/or BSO if unsure. Once completed the user and their confirmation will be added to the list of authorised lab users by the BSO. The status and content of the SOP framework is the responsibility of the Biological Safety Committee and, when relevant, the Genetic Modification Safety Committee. These committees should be informed of the nature of the work any users of the laboratory wish to undertake. The immediate point of contact is the BSO.

TODO It's not clear whether the inductee should notify the Committees or the BSO prior to commencing work. It seems like the inductee should notify the BSO, and it is the BSO's responsibility to make sure the cttees are aware. Either way, clarification required.

For those starting new projects in the laboratory, full details of the requirements of principal investigators, in particular risk assessments for new projects, can be found in LBL06001, Project Risk Assessment Procedure for Lab Users Proposing New Projects.

6.2 PPE, Biosafety and Contamination Risks

The kind of biological organisms with which we typically work in the laboratory include i) non-pathogenic strains of E. coli (such as DH5 alpha), ii) cellulose producing gram-negative bacteria such as Gluconacetobacter, iii) non-toxic Dinoflagelate species, iv) brewer’s yeast (Saccharomices cereviscae) and, v) algal microorganisms from non-toxic environmental samples.

These organisms do not themselves require working containment level 1 (CL1) standards, however it is still considered good laboratory practice to minimise exposure of any laboratory user to direct contact with them and to prevent any biomaterial from being unintentionally carried out of the laboratory. Further, those organisms which do fall into the classification of CL1 requirements, such as those strains genetically modified by transformation (for example, E. coli by heat shock method, and Gluconacetobacter by electroporation), should be treated with extra care. Any surplus material must be destroyed in the kill bin, and all other waste must be autoclaved. Personal Protective Equipment (PPE) suitable for working with these kinds of organisms is provided and consists of lab coats, goggles and gloves. The BSO will indicate the location of PPE to the new inductee. If suitable PPE is not available, the BSO should be informed immediately before any practical work is commenced. To ensure that any contamination of genetically modified biomaterial and microorganisms does not occur, laboratory users should be aware of the correct handling and disposal procedures required. They should be made aware of typical requirements during laboratory safety induction, and this knowledge should be reinforced during individual project risk assessments. Generally, all microorganisms should be handled while using gloves, eye protection and protective clothing to prevent material coming into contact with the user’s skin or eyes. This is both to protect the user and to eliminate the risk of transporting biomaterial out of the laboratory after work is completed. Gloves can be disposed of in the bins provided. Containment Level 1 biomaterial should be deactivated before disposal by autoclaving and immersion in bleach in the ‘kill bin’ underneath the sink. The deactivated waste in the kill bin can then be autoclaved and then diluted further and disposed of down the drain. These handling and disposal procedures are explained later in the SOPs but are highlighted here to communicate the important message that biosafety and protection of users of the laboratory is of the highest importance.

6.3 Microwave

The microwave is a familiar device to probably all laboratory users and the standard precautions apply when using it. In particular, the handling of hot liquids and glassware may present the risk of burns and as such thermally resistant gloves are provided. Notably, one of the primary uses of the microwave is heating of agarose for the casting of electrophoresis gels. Users should be aware that prior, possibly less careful, microwave operators may have inadvertently contaminated the microwave with biological material and/or hazardous chemicals (as described below), and as such operation of the microwave using gloves and goggles is recommended in any case.

More information on microwave usage is available in LBL04008, Using the Microwave.

6.4 Spectrophotometer

The spectrophotometer is a device that measure the absorbance of a given liquid sample at various wavelengths of light. A full description of its usage is given in LBL04001, Operating the Spectrophotometer. Users should be aware that this system can generate light of harmful intensities in both ultraviolet and infrared wavelengths. While most of the optical path and the bulbs required to generate this light is contained within the system, it is beamed into the sample measurement compartment of the device. As such the spectrophotometer should be operated only with the sample measurement compartment fully closed.

6.5 PCR Machine

The Polymerase Chain Reaction (PCR) machine, also known as a thermal cycler, is used to amplify specific sections of template DNA either for diagnostic or molecular cloning purposes. The heating block and underside lid of this machine will typically reach temperatures in the region of 95 to 100degC in a typical amplification program and caution should be exercised when placing and removing samples. Information regarding the current state of the machine including current program and temperature is displayed on the LCD on the front panel of this machine. Full information regarding the use and programming of the Techne thermal cycler and its use in various procedures can be found in LBL07004, Setting up and running PCRs, and the technical documentation. [docs]

6.6 Electrophoresis Power Supply Unit

The Power Supply Unit (PSU) used to supply a current to the electrophoresis tank (described below) is capable of producing dangerously high voltages and currents. It is typically set to the levels required for DNA electrophoresis in 100mL 1 - 2% w/v agarose gels, i.e. between 60 and 120 volts DC. Caution should be exercised when plugging in and handling electrodes due to risk of electric shock. Use of a residual current device is recommended when using this or any other electrical device in the laboratory that may pose a risk to operators from electric shock. This instrument is also often used in conjunction with Ethidium Bromide based procedures (see below), and may therefore be contaminated. It must be operated using gloves.

6.7 Electrophoresis tank and Ethidium Bromide area

The electrophoresis tank is located within the electrophoresis/Ethidium Bromide area of the laboratory bench. It is advised any work carried out within this area is manipulated separately from any work done in other areas, due to the risk of contamination with Ethidium Bromide. Any consumables used when handling Ethidium Bromide, such as gloves and tips, should be disposed of before anything outside of this area is handled to prevent contamination of the wider lab area with the potentially toxic chemicals used in DNA staining. Separate pipettes designated for Ethidium Bromide work and labelled as such should only be used for molecular biology work in this area, and these pipettes should not be used anywhere else. A full description of how to set up and run agarose gels for DNA electrophoresis is described in the SOPs [SOP].

6.8 UV illuminator

Gel electrophoresis separates DNA by size within an electric field. Separation of varying sizes of DNA fragments within an agarose gel allows visualisation of the various sizes of DNA fragment contained within a sample when nucleic acid binding stains are used. The typical stain used is Ethidium Bromide which is a fluorophore that is excited within the UV spectrum (with excitation maxima under 300nm) and emission within the visible spectrum. This presents two main risks to the user: i) As a DNA intercalating agent Ethidium Bromide is potentially carcinogenic and should never be allowed to come in contact with the user’s skin. Caution should therefore be applied throughout the entire DNA electrophoresis procedure from gel preparation, through electrophoresis and then subsequent gel visualisation. All parts of the procedure must be performed in the electrophoresis/ethidium bromide area of the laboratory bench. ii) UV light can be damaging to exposed surfaces of the body and, especially, to the eyes. In extreme circumstances, or under prolonged use, this can lead to carcinomas or eyesight damage. Eye protection must, therefore, be used by all people present within the laboratory when UV gel visualisation is taking place. Suitable glasses are available and will be indicated by the BSO. Further, users of the illuminator must ensure that PPE prevents any UV light from reaching exposed skin. Users should aim to minimise the time in which the UV light source is switched on. [SOP for gel vis]

6.9 HEPA flow cabinet/area

The HEPA-filtered laminar flow unit allows us to work in sterile air in order to prevent contamination of our work, such as petri dishes and broths, with other microorganisms. Proper usage of the laminar flow area is described in the SOPs. It should be noted that the laminar flow functions in such a way as to protect the user’s work rather than the user, and so caution must still be exercised by the user when maintain the aseptic conditions of materials used within the flow area, and in disposal of consumables to assist in containment of biomaterial. For more information on aseptic technique, see LBL04005, Operating the Laminar Flow Unit for asceptic sample handling.

6.10 Autoclave

The autoclave is used to destroy any potential microorganisms that might contaminate media, reagents and consumables to be used aseptically in micro- and molecular biological procedures. High temperature and pressure is used to kill contaminants. The high temperature and pressure is accompanied by a vacuum cycle. Users should familiarise themselves with the instructions for use [REF] before working with the autoclave. The main risks to the user are heat of the metal pressurised unit during sterilisation and any vented steam. It is recommended that the autoclave and contents are allowed to cool for a while before handling. The correct procedures for using the autoclave/steriliser to prepare media and destroy GM waste are described in the SOPs LBL04002 (Using the Autoclave for Media Preparation) and LBL04006 (Using the Autoclave for Waste Deactivation).

6.11 Sink area

The sink area should remain clear. All glassware should be kept clean and out of the way.

The sink is connected to the municipal drain. No biological material should be disposed of in the sink unless it has been inactivated appropriately (see LBL04006 for autoclave-based inactivation, or LBL04007 for kill-bin-based disposal). Dangerous reagents and chemicals, in particular Ethidium Bromide, should not be poured down the sink. If there is any doubt about whether a particular chemical or reagent can be disposed of safely, consult the BSO.

6.12 Chemicals and Storage

The chemicals we use have individual storage requirements, detailed in the list of chemicals [REF]. Before using any chemical, laboratory users must familiarise themselves with the material safety data sheet (MSDS) for that chemical. A list of MSDSes is available at [REF] [SOPs appendix A]

6.13 Incubator

The incubator is typically used for the culture of mesophiles, and, as such, does not operate at dangerous temperatures. Care should be taken when moving samples to and from the incubator as per handling biocontaminants mentioned previously. Many procedures described later in the SOP documentation indicate the need for incubation. Please refer to LBL04004, Using The Incubator, for more information.

6.14 Centrifuges

The laboratory has a number of centrifuges available for use. The Jouan is a larger device which can take 50ml tubes and larger containers if fitted with buckets and can spin up to 10,000 rpm (although documentation and instrument panel indicate higher rpm may be possible this particular unit appears to have a limit of 10,000.) The Henle and MSE microcentaur can spin up to higher rpm with smaller samples. These centrifuges will not operate in their normal state without a closed lid, preventing the user from coming into contact with the moving parts. There are also two blood centrifuges which spin in the region of 1000 to 2000 rpm. These centrifuges do not have safety interlocks, so it is possible to start these centrifuges with their lids open. This is highly dangerous, as there is a risk both of exposure to machinery moving at dangerous speeds, and of spray from poorly-sealed liquid samples. Users of these centrifuges must ensure that the lids are closed prior to operation. All centrifuges should be cleaned after use for the purpose of biosafety, containment and equipment maintenance. For more information on the use of the centrifuges, refer to LBL04003, The BioLab Centrifuges and their usage.

6.15 Refrigerator, Freezer and Sample Storage

The refrigerator maintains samples and reagents at 4degC. Typically the refrigerator stores bacterial plates and reagents for transformation. Due to the presence of biomaterial in the refrigerator, caution should be exercised to prevent contamination of the user. The freezer contains longer term bacterial stocks and enzymatic reagents, maintaing a temperature of at least -20degC. Similar caution should be taken when using the freezer to avoid personal contamination. Specific details for the handling of the various chemicals and reagents stored in the freezer and refrigerator can be found in the chemicals and storage list [SOP appendix A]. Specific SOPs also cover the storage and recovery of frozen bacterial stocks and procedures requiring the usage of frozen and refrigerated reagents. [SOPs]

7. Resources

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