1. INTRODUCTION
1.1 Aim
1.2 Document Layout
1.3 Deciding which CL Applies
2. PROCEDURES
2.1 Emergencies, Spills and Incident/Accident Reporting
2.2 Risk Assessment and Training
2.3 General Operating Procedures
2.4 Waste Disposal and Disinfection
2.5 Safe Sample Storage and Transport
2.6 Validation, Maintenance, Testing and Monitoring
1 Introduction
1.1 Aim
The following procedures
observe the principles of GMP and GOSH and are derived from guidance published
by the ACDP, SACGM and University Safety Services. They aim to provide comprehensive
advice on how to achieve and maintain CL1 and CL2 when working with micro-organisms,
including those which have been genetically modified, within SLS laboratories. These procedures also apply to any material that is, or
may be, infected/contaminated with micro-organisms, for example, human or animal
blood and tissue.
1.2 Document Layout
Procedures are divided into six sections:
1. Emergencies, Spills and Accident/Incident Reporting;
2. Risk Assessment and Training;
3. General Operating Procedures;
4. Waste Disposal and Disinfection;
5. Safe Sample Storage and Transport;
6. Validation, Monitoring, Testing and Maintenance;
each comprised of one or more sub-sections. Each sub-section firstly stipulates the basic procedure that must be applied to all work involving micro-organisms, which in many instances will be sufficiently rigorous to satisfy CL1 and CL2 requirements. However, in certain instances, it is not practical to apply CL2 when only CL1 is strictly required. In such cases the basic procedure is designed to achieve CL1 and is supplemented by additional or special measures necessary to achieve CL2. Special measures replace the basic procedure whereas additional measures supplement it. Such measures are clearly indicated.
To reduce scope for error, every effort has been made to apply the same procedures to both the general laboratory and the TC suites. However, certain Procedures will apply only to the general laboratory and some only to TC suites. Such procedures will be clearly indicated; all others apply to both areas.
Certain general procedures, as opposed to procedures solely applicable to work at CL1 or 2, are not reiterated here, but links to the relevant documents are provided.
Prior risk assessment will clearly determine which CL applies to any given work activity. You must complete a risk assessment before undertaking any work activity on SLS premises.
As a general guide, CL2 applies to work involving:
1. Human
pathogens classified by the ACDP into Hazard Group 2 (Note 1).
2. Micro-organisms, including those which have been genetically modified,
not classified by ACDP but shown by risk assessment to present a hazard equivalent
to an ACDP Hazard Group 2 pathogen (Note 1).
3. Micro-organisms, including those that have been genetically modified, shown
by risk assessment to present a hazard to the external environment e.g. plant
or animal pathogens.
4. Viral vectors capable of infecting human cells, regardless of whether they
are replication defective or otherwise attenuated relative to the wild type.
Risk assessment may show that CL1 is required but it is SLS policy to apply
CL2.
5. Blood, tissue or cell culture known or suspected to be infected with any
of the above (Note 2).
6. Finite or continuous cell lines of human or simian origin not fully authenticated
or characterised.
7. Primary cells derived from blood, lymphoid or neural tissue of human or
simian origin (Note 3).
8. Human blood and tissue not falling into category 5 (Note 3 and 4).
Note 1: The ACDP Approved List of Biological Agents is published on the HSE web site and gives adivce on risk assessment and definitions of the official Hazard Groups.
Note 2: This category includes chicken egg material.
Note 3: This includes screened and unscreened blood/tissue/cells.
Note 4: You must consult the SLS Health & Safety Information Officer before
undertaking any work activity involving the use of human blood.
2 Procedures
2.1 Emergencies, Spills and Incident/Accident Reporting
2.1.a Fire
In the event of a fire the SLS Fire Procedure must be followed.
2.1.b Personal Injury
In the event of personal injury the SLS First Aid Procedure must be followed.
2.1.c Spill
In the event of a spill SOP number 62 must be followed.
2.1.d Accident/Incident Reporting
In the event of any accident or incident the SLS Accident & Incident Reporting Procedure must be followed.
2.2 Risk Assessment and Training
2.2.a Risk Assessment
As per SLS Risk Assessment arrangements.
2.2.b Training
As per SLS Health & Safety Training arrangements.
2.3 General Operating Procedures
2.3.a Organisation
2.3.b Hygiene
2.3.c PPE
2.3.d Working Practices and Safe Use of Equipment
2.3.e Disinfection of Work Surfaces and Equipment
2.3.f Treatment of Contaminated, Reusable Glass/Plastic Ware
2.3.g Fumigation of Equipment
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2.3.a Organisation
- Writing rooms directly off lab bays may be designated as laboratory or non-laboratory areas at the discretion of the Group Leader. If designated as a laboratory area they cannot be used for storage of outdoor clothing and bags nor storage/consumption of food/drink.
- Do not leave or wedge laboratory doors open.
- Keep laboratory surfaces clean, tidy and well organised.
Additional CL2 Measures:
- Lab access must be restricted to authorised personnel.
- A biohazard sign must be permanently displayed on the CL2 Facility doors.
- Writing rooms directly off CL2 lab bays are considered laboratory areas. This means storage of outdoor clothing/bags and storage/consumption of food/drink is prohibited. However, they may be designated as clean lab areas kept solely for writing up/computer work. No chemicals or biological materials are to be handled or stored in these clean lab write-up areas. PPE is to be removed and hands washed before entering clean lab write-up areas.
2.3.b Hygiene
Good Laboratory Practice details standard laboratory hygiene measures.
Additional CL2 Measures:
- Hand-washing sinks must have elbow operated taps.
(updated 4/2/2020)
- Lab coat wearing is mandatory in all areas bar the clean write-up areas described above. Lab coats should be side or back fastening with high collar and elasticated cuffs (i.e. Howie style). Lab coats must be removed upon leaving the laboratory area and stored on the dedicated pegs provided. The CTS Technician will collect lab coats for laundry as required. If a lab coat becomes contaminated, seal in an autoclavable bin-liner and inform the CTS Technician. It will then be autoclaved before laundering. Keep a separate lab coat for wearing only in the TC suites. Remove before leaving the TC facility and store in the TC anteroom on the dedicated pegs provided. TC lab coats will be autoclaved before laundering as a matter-of-course.
- Areas of the body not covered by a lab coat should be fully covered by clothing and footwear.
- If skin contamination presents a risk of infection wear appropriate disposable gloves. Remove contaminated gloves immediately, dispose of as solid waste and replace with a fresh pair. Gloves should be removed before handling items likely to be touched by persons not wearing gloves, e.g. telephone, paperwork.
- Wearing of safety glasses is mandatory in all areas bar the clean write-up areas described above.
Additional CL2 Measures:
- Dark blue is the designated colour for CL2 Facility lab coats.
2.3.d Working Practices and Safe Use of Equipment
- All procedures should be carried out so as to minimise the production of aerosols.
- Use of sharps is prohibited unless essential
- When using shaking incubators: anchor flasks/tubes securely to prevent spills/breakage; do not overfill flasks; ensure maximum rpm for flask size is not exceeded.
- When using centrifuges: adhere to the manufacturers instructions; use tubes certified by the manufacturer as suitable for low/high/ultra speed centrifugation; observe maximum fill volumes and spin speeds; do not use visibly damaged tubes or rotors; check all o-rings are greased and in place to ensure leak-proof seals.
Additional CL2 Measures:
- Use of an MSC is mandatory if there is risk of infection via the aerosol route. MSCs must be class I or II and bear the biohazard symbol. Users must receive training before first use.
- If working outwith
an MSC all procedures must be performed so as to minimise, the production
of aerosols:
- Avoid vortexing, sonicating, rapid pouring and use of homogenisers on the open bench.
- Use aerosol resistant pipette tips and plugged pipettes.
- Use pipettes slowly and carefully.
- Eject pipette tips carefully and directly into sharps-safe container.
- During centrifugation procedures use aerosol containment tubes, canisters and/or rotors in accordance with the centrifuge manufacturer's instructions. Note: where there is risk of infection by aerosol inhalation, aerosol containment vessels must only be opened within an MSC.
- Ensure incubators are labelled with a large biohazard sign and all culture vessels are labelled with your name and the nature of the sample.
- When using shaking incubators, ensure tubes are capped and flasks are plugged and topped with foil. Wait until the culture stops swirling before removing the cap/foil. Look carefully through the viewing panel to check for spills/breakages before opening an incubator. If a spill has occurred, allow time for aerosols to settle before opening the door.
- When using a constant temperature/CO2 incubator use culture vessels that will prevent leaks/spills if at all possible, e.g. filter cap flasks. Otherwise, place culture vessels in a tray that will contain any leaks/spills.
2.3.e Disinfection of Work Surfaces and Equipment
After working with micro-organisms, thoroughly spray work surfaces
with 1% Virkon solution, leave for ten minutes then thoroughly wipe down with
70% ethanol to remove any residue.
Notes: Before disinfecting metal items check the Virkon web site for compatibility
data. Do not spray sensitive equipment with Virkon - consult your Lab manager
or BSO for advice. Do not use Virkon solution if it is more than five days
old or if the pink colour has faded: it may not be effective! Disinfection must
be validated - see section 2.6.a. and consult
your BSO for specialist advice .
2.3.f Treatment of Contaminated, Reusable Glass/Plastic Ware
- Autoclavable items must be placed with liquid waste containers in the designated collection are for collection by the CTS Technician. There must be no significant amount of waste material present (e.g. no pellets or volume of liquid), only trace contamination. Before autoclaving any item, check that it is fully autoclavable.
- Non-autoclavable items must be disinfected by fully submerging in 1% Virkon overnight. Safely contain the disinfecting items and, to avoid accidental removal or confusion over disinfection time, clearly label the container with the nature of the sample, your name, the date and time. Once disinfected, rinse thoroughly with water before sending into the normal wash-up stream. Notes: Before disinfecting metal items check the Virkon web site for compatibility data. Do not use Virkon solution if it is more than five days old or if the pink colour has faded: it may not be effective! Disinfection must be validated - see section 2.6.a. and consult your BSO for specialist advice.
Additional CL2 Measures:
- Ensure the container used for disinfection is clearly biohazard labelled and stored in a demarcated area
2.3.g Fumigation of Equipment
Microbiological safety cabinets must be fumigated: prior to
servicing or repair that involves accessing internal areas/parts; if the MSC
has been used for CL2 activities since its immediately previous fumigation;
before final disposal; in the event of a major spill of Hazard Group 2 material;
and if usage changes from CL2 to CL1.
Note: you must inform the SLS Health and Safety Information Officer of MSC fumigation, preferably one week in advance and 3 working days in advance as a minimum.
2.5 Safe Sample Storage and Transport
2.5.a
Storage of Samples
2.5.b Transport Within the Building
2.5.c Transport Out With the Building
2.5.d Consignment by Post
2.5.a Storage of Samples
All
samples must be stored securely and clearly labelled - with the nature of the
sample and a contact name - so as to prevent spillage, loss, theft, access by
unauthorised personnel or accidental removal. Samples stored in liquid nitrogen
cryo-stores must be contained in vials/tubes specifically designed for the purpose.
All other samples should be stored in appropriate robust, leak-proof containers.
An effective mechanism for identifying, tracking and auditing stored samples
of micro-organisms must be in place, e.g. up-to-date, detailed inventory of
–80 freezer or liquid nitrogen cryo-store racks. Records must be available
for inspection upon request.
Additional CL2 Measures:
- Samples stored in fridges or freezers must be doubly contained in robust, leak-proof containers.
- All storage vessels (e.g. fridges, freezers, liquid nitrogen cryo-stores) must bear the biohazard symbol.
- If the storage vessel is not housed in a secure building or facility it must be kept locked with only authorised personnel having access to the key.
- Samples kept in liquid nitrogen cryo-stores must be stored in the vapour phase to eliminate the risk of tube explosion upon initial warming.
2.5.b Transport Within the Building
It
is preferable to transport samples in robust, leak-proof containers. Containers
should be labelled with the nature of the sample and a contact name. Containers
that do not fulfil these criteria, e.g. glass flasks/bottles, must be transported
in a plastic tub on a trolley with a retaining lip or in a deep 'bucket' trolley. Do not overfill vessels that cannot be sealed
for transport, e.g. glass conical flasks.
Special CL2 Measures:
- Samples must be doubly contained during transport outwith the containment facility and clearly labelled with the nature of the sample, a contact name and the biohazard symbol.
- The inner container must be robust and leak-proof. The outer container must robust, leak-proof and contain enough absorbent material to absorb the total volume of sample should the inner container leak.
2.5.c Transport Outwith the Building via Road
- Samples must be doubly contained during transport. The inner container must be robust and leak-proof. The outer container must be robust, leak-proof and contain enough absorbent material to absorb the total volume of sample should the inner container leak.
- The outer container must be sealed during transport and must clearly display the nature of the sample, a contact name, work address and telephone number in case of loss in transit.
- The individual transporting the package must be trained in how to deal with spillage or loss.
- The preferred option is to arrange for Safety Services to transport the package. If this is not possible, a private car, a taxi, or public transport may be used. If using a private car the driver must check that their insurance covers the use of the vehicle for business purposes.
Special CL2 Measures:
- The UBSA should be consulted before the package is transported. Give a full and accurate description of the sample. In certain cases additional measures may be required.
- Inner container must also be clearly labelled, as above, and both containers must bear the biohazard symbol.
- Use a reputable courier, e.g World Courier, and adhere to their requirements for packaging and labelling of biological material. Ensure they are fully aware of the hazard status and nature of the sample.
- If sending material abroad, the sender must check the regulatory requirements of the country of destination.
- Contact Safety Services for further advice on sending micro-organisms within or outwith the UK.
- If shipping GM or hazard Group 2 material, you must consult the UBSA beforehand.
2.6 Validation, Maintenance, Testing and Monitoring
2.6.a
Disinfection Validation
2.6.b Autoclave Testing and Maintenance
2.6.c Maintaining PPE
2.6.d Inspections, Audits and Continual Monitoring
2.6.e MSC Testing and Maintenance (TC Only)
2.6.f Negative Pressure Testing (TC suites only)
2.6.g Environmental Monitoring
2.6.a Disinfection Validation
Virkon is
the disinfectant of choice but, where it is shown to be ineffective, other disinfectants
may be used. The chosen disinfection method must be proven to be effective under
the specific conditions of use. If used in accordance with the manufacturers
instructions, i.e. adhering to specified concentration and contact time, and
if documented as effective against the contaminating micro-organism(s) under
these conditions, no further validation is required. If these criteria are not
fulfilled, and especially in cases where disinfectant is used to treat volumes
of culture fluid, validation by experimental means is required. An outline protocol
for disinfection validation is shown below.
Note: Virkon efficacy data: originally published by DuPont; published by Lanxess.
Outline Protocol for Disinfection Validation
The composition of the culture fluid, cell density, contact time and final concentration must be documented. 5-log reduction (99.999% kill) must be achieved.
- Grow up micro-organism under the typical experimental conditions.
- Add disinfectant to a known culture volume to give the desired final concentration.
- Incubate at room temperature for specified time period.
- Take a sample from the incubation mixture
- Eliminate traces of disinfectant by washing cells into fresh culture medium.
- Attempt to grow any surviving micro-organisms on plates or in liquid culture. Note – for viruses attempt to grow in host cell line.
- Count survivors and calculate % kill.
2.6.b
Autoclave Testing and Maintenance
Click here.
2.6.c Maintaining PPE
Users are
required to routinely check their PPE (e.g. lab coat, safety glasses) and keep
it in good order. Defective PPE must be repaired or replaced immediately. Laboratory
Managers are required to ensure the appropriate PPE is readily available and
keep an inspection record for non-standard PPE, e.g. that used in Liquid Nitrogen
facilities.
2.6.d
Inspections, Audits and Continual Monitoring
(amended 12/4/17)
- Safety Inspections are carried out regularly to ensure health & safety policy & procedures are being followed and that the required risk assessments and training records are complete and up-to-date. Inspections will be timetabled and inspection teams selected by SLS Safety. Inspection team members will be selected from SLS Health & Safety personnel, Lab Managers and Senior Management. Inspection reports will be made available to all SLS personnel via the SLS Safety website and brought to the attention of the relevant senior manager(s).
- Audits performed by an external, independent body will be arranged by the University Safety Services as they deem necessary.
- All personnel are expected to continually monitor safety standards and compliance with Health & Safety Policy & Procedures within their work area and report problems and non-compliance to their Line Manager and SLS Safety.
2.6.e
MSC Testing and Maintenance (TC Only)
MSCs
are serviced and operator protection (KI) tested on an annual basis by a reputable
service provider. Certificates of conformity to the required standard must be
displayed on each cabinet. SLS Safety are responsible
for arranging the servicing schedule, ensuring bio-decontamination is
carried out prior to testing and issuing and keeping a copy of the certificates
of conformity to the relevant standard. Users are required to perform a visual check on all alarms and
indicators before each use and report any defects immediately to their Lab Manager.
Note: If an MSC is moved to a new location, or equipment in a room containing
a cabinet is significantly re-arranged, to the extent where it may affect the
airflows within the room, the cabinet must be KI tested before reuse to ensure
operator protection has not been compromised.
2.6.f Negative Pressure Testing (TC suites only)
(updated 12/4/17)
In the CL2 TC suites, all work is carried out within microbiological safety cabinets, therefore, maintaining a net inward flow of air is not essential.
2.6.g Environmental Monitoring
Environmental monitoring may be undertaken in areas where CL2 activities that could produce aerosols are being carried out on the open bench, i.e. outwith an MSC. This scenario is most likely to arise in labs where pathogens that are transmissble via ingestion, as opposed to inhalation, are in use. Aerosols may not be the primary concern but they can disperse throughout the lab and contaminate surfaces with viable microorganisms that may be inadvertantly picked up and carried out of the lab. Environmental monitoring can be used to demonstrate that aerosol production/dissemination is being effectlvely controlled.