BioLab Usage Guidelines

Template:Template As specified in the BioLab Rules the following advice must be followed when working in the lab.

General Safety

Worktops should be left as clear as possible after work has been completed.

All containers should be labelled with contents, date and initials of the lab user. Unlabelled containers are a safety concern.

Any incidents of spillage, equipment malfunction, or injury should be recorded in the day book and reported to the BSO.

Before starting a project lab users should:

• familiarise themselves with the material safety data sheet (MSDS) of any reagent to be used in a project,
• be aware of all the SOPs that address the kind of procedures to be carried out in a project,
• be aware of whether the project involves working with GMOs, and
• consult the BSO if unsure of the safety requirements and skill level necessary to carry out a project.

All SOPs can be found in the physical copy of the lab manual located in the lab and online at BioLabSOPs.

A list of MSDSes is available in SOP Appendix A: BioLab Chemicals MSDS Catalogue LBL0A002

Chemicals and Storage

The chemicals used and stored in the lab have individual storage requirements, detailed in the SOP Appendix A: BioLab Chemical Storage LBL0A001.

Before using any chemical, laboratory users must familiarise themselves with the material safety data sheet (MSDS) for that chemical.

A list of MSDSs is available in Appendix A: BioLab Chemicals MSDS Catalogue LBL0A002

Use of PPE

Personal Protective Equipment (PPE) suitable for working with reagents and low risk micro-organisms is provided. The location of all PPE is indicated during the lab safety induction.

Lab coats should be worn when working with micro-organisms or reagents.

Disposable gloves should be worn when indicated by the MSDS of a reagent being worked with and during any work with micro-organisms.

Protective eyewear should be worn when indicated by the MSDS of a reagent being worked with and during any work with micro-organisms.

If suitable PPE is not available, the BSO should be informed immediately before any practical work is commenced.

Working with Micro-organisms

In accordance with lab rule 5, before working with any micro-organisms, users must consult appropriate databases to ensure that the biological agent is unlikely to cause human disease and therefore considered to be in Hazard Group 1.

The Approved List of Biological Agents should be consulted to verify that the biological agent is not considered to be in Hazard Group 2 or above. Additional databases should be consulted to decide if the biological agent is unlikely to cause human disease.

Resources for determining hazard group:

• Approved List of Biological Agents: (http://www.hse.gov.uk/pubns/misc208.pdf)
• DSMZ Catalogue of microorganisms (http://www.dsmz.de/catalogues/catalogue-microorganisms.html)

If the user is unsure of the Hazard Group of a biological agent, the BSO should be sought before any work with the agent commences.

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 are considered to be Hazard Group 1 and do not themselves require working containment level 1 (CL1) standards, however, in addition to lab rules 2 and 3, the following good microbiological practice (GMP) should be employed when working with any micro-organisms.

Wear gloves if skin on the hand is broken, if a rash is present, and when handling biological waste. Remove gloves before leaving the lab, touching the face, keyboards, or control panels. Gloves should be disposed of in the bins provided.

Wear protective eyewear during procedures in which splashes of microorganisms or other hazardous materials is anticipated to prevent material coming into contact with the eyes.

Ensure the work area is clean and uncluttered to reduce the chance of cross-contamination and inadvertent exposure to biohazards.

To avoid the risk of ingesting contaminated material do not mouth pipette or apply cosmeticis in the lab.

Procedures should be performed in a manner that minimizes the creation of aerosols.

Clean and decontaminate work surfaces with a suitable disinfectant at the end of the day and after any spill of potentially hazardous materials.

Biohazardous material must be clearly marked with a biohazard symbol.

Special GMO considerations

In accordance with lab rule 6, users must be aware of what constitutes a project involving genetic modification and therefore whether such a project requires Containment Level 1 (CL1).

Below are definitions reproduced from The Genetically Modified Organisms (Contained Use) Regulations 2014 Guidance on Regulations issued by the Health and Safety Executive:

Genetic modification: Means any alteration of the genetic material of an organism (ie DNA or RNA), which does not occur naturally (by mating or recombination) and which has been achieved through one of the techniques set out in Part 1 of Schedule 2. The techniques listed are examples and are indicative of the types of alterations that fall within the Regulations. The requirements of the Regulations (eg risk assessment, application of control measures) apply to the activity in which GMOs are created, used or disposed of rather than the techniques themselves.

These techniques involve introducing and incorporating new combinations of genetic material (whether derived from an existing organism or synthetically made) into a recipient organism in which they do not naturally occur. The introduced genetic material must be capable of stable incorporation and/or continued propagation in the recipient organism. Techniques considered to be genetic modification include:

(a) any technique which alters the genetic material in an organism using a method that does not occur by natural mating or recombination (eg synthetic generation of artificial chromosomes in yeast);

(b) introduction of foreign or synthetic genetic material into an organism via transfection, recombinant bacteriophage transduction (eg to make gene libraries), transformation, particle bombardment or other gene delivery systems (eg liposomes);

(c) gene deletions or the insertion of multiple copies of a gene in an organism count as genetic modification if they are brought about using any listed technique or other artificial method;

(d) stable introduction of synthetically generated DNA or RNA (eg ‘biobricks’) into an organism;

(e) techniques that involve directly introducing heritable genetic material (eg particle bombardment of plant tissues, directly injecting naked DNA into an animal and liposomes) only where the introduced genetic material is intended to be incorporated into the organism’s genetic material in a stable way.

—Paragraph 20-21, The Genetically Modified Organisms Contained Use Regulations 2014 Guidance on Regulations

Techniques that are not considered to be genetic modification include:

(a) organisms generated using methods based on natural mating or recombination;

(b) somatic cell nuclear transfer (‘cloning’) provided no GM material is present and the donor/recipient organisms are able to interbreed;

(c) artificial transfer of pollen from one flower to another (considered to be natural fertilisation);

(d) hybrid or reassortant viruses generated by natural recombination or transencapsidation during co-infection of a cell;

(e) DNA vaccination, where naked or synthetic DNA is introduced into animals to elicit an immune response against antigens encoded by that material, with no intention of stable integration.

—Paragraph 22, The Genetically Modified Organisms (Contained Use) Regulations 2014 Guidance on Regulations

If the user is unsure of whether a proposed project requires CL1, the BSO should be sought before any work on the project commences.

A user must have an approved risk assessment for any work involving genetic modification before commencing this work. More information on the process of risk assessment and approval can be found in SOP LBL06001 Project Risk Assessment Procedure for Lab Users Proposing New Projects. A seperate risk assessment must be performed and approved for transporting any GMO outside the laboratory (details in SOP LBL06002 Risk Assessment Procedure for Lab Users Proposing Transport of GMOs Outside the Lab).

CL1 activities must follow the procedures set out in LBL05001 Containment Level One For BioLab Users and the further procedures set out in the approved risk assessment.

In general this includes:

• following GMP as set out in section X (Working with micro-organisms) of these guidelines when working with micro-organisms,
• all biomaterial and waste that was in contact with a genetically modified micro-organism (GMM) must be deactivated by autoclaving before disposal (the autoclave must have passed the deactivation test that day),
• taking care to prevent the dispersal of the GMO outside the laboratory.

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, maintaining 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]

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.

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.

Spectrophotometer

The spectrophotometer is a device that measure the absorbance of a given liquid sample at various wavelengths of light.

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.

More information on spectrophotometer usage is available in LBL04001, Operating the Spectrophotometer.

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. 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.

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.

More information on usage of the Techne thermal cycler is available in LBL07004, Setting up and running PCRs, and the technical documentation. [docs]

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.

More information on electrophoresis PSU usage is available in LBL07003, Gel preparation and electrophoresis.

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 available in LBL07003, Gel preparation and electrophoresis.

More information on working with ethidium bromide is available in LBL07005, Handling and Storage of Ethidium Bromide (EtBr).

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:

a) 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.

b) 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.

More information on working with the UV illuminator is available in LBL07006, UV Illumination of electrophoresis gels.

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.

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.

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.

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.

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.

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.

Please refer to LBL04004, Using The Incubator, for more information.

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. 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.

Due to the high rotation speed of the centrifuge arms, it is essential that loads be balanced during operation of the centrifuges. When loading a centrifuge, ensure that samples are placed in such a way as to create a balanced load (it may be necessary to use blanks in order to achieve this). Failure to do so could result in serious damage to the centrifuge and possibly cause injury in the case of a catastrophic failure.

For more information on the use of the centrifuges, refer to LBL04003, The BioLab Centrifuges and their usage.

Resources

BioLabSOPs

Approved List of Biological Agents: (http://www.hse.gov.uk/pubns/misc208.pdf)

DSMZ Catalogue of microorganisms (http://www.dsmz.de/catalogues/catalogue-microorganisms.html)

HSE GMO Regulations index page (http://www.hse.gov.uk/biosafety/GMO/index.htm)

The Genetically Modified Organisms (Contained Use) Regulations 2014 Guidance on Regulations (http://www.hse.gov.uk/pubns/books/l29.htm)

The Genetically Modified Organisms (Contained Use) Regulations 2014 (http://www.legislation.gov.uk/uksi/2014/1663/contents/made)