Risks
In addition to those safety challenges commonly encountered in chemical laboratories, procedures undertaken in microbiological laboratories - indeed all procedures involving micro-organisms regardless of laboratory type - pose particular safety problems. Pathogens must be handled with great care in order to avoid infection of staff, the general public and animals outside the laboratory.
Microbiological hazards are particularly insidious because of the microscopic size of the organism.
The safest procedure is to regard all micro-organisms as potential pathogens and treat them accordingly.
Protecting people from infection
Infection can result from ingestion, inhalation or skin penetration. In particular, staff or students having little or no microbiological training should not be exposed to situations in which they may not appreciate the potential hazards.
Non-laboratory workers such as cleaners and tradesmen should be given special instruction if they are to come into contact with this class of hazard. No one should be working in a microbiological environment without knowledge of recommended practices and procedures.
Preventing cross-contamination of results
Prevention of cross-contamination or contamination with adventitious micro-organisms is important since this may nullify experimental procedures and lead to erroneous results. Such a situation may result in incorrect treatment of patients or modified techniques.
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Warning signs
A biological hazard must be clearly indicated by standard biological warning signs giving the type and degree of risk and the person responsible. Immediately adjacent to the symbol, a sign shall also be displayed stating: Danger - infectious material.
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Separate areas
Separate areas should be set aside for:
preparation of media
holding of materials
sterilisation
storage of sterile articles
collection of specimens from patients
receipt of samples – spill trays should be provided.
Animal rooms must be segregated from laboratories and should contain separate areas for infected animals, for non-infected animals and post-mortems.
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Protective clothing
Protective clothing should be worn in microbiological laboratories, and gowns or coats removed before leaving the laboratory for common rooms, office areas or home.
Protective gloves should be worn in highly infective situations, and personnel should disinfect their hands before and after using gloves, as minute holes may allow entry of micro-organisms.
Elbow or foot operated taps should be available for washing as well as emergency showers.
Wounds and infections provide excellent routes for further infections. Any cut or abrasion should be treated immediately and covered with a waterproof dressing. Any infections, particularly of the respiratory or alimentary tracts or hand wounds, must be reported immediately.
Details: Australian Standard AS2243.3. Safety in Laboratories Part 3 – Microbiology. See the Standards Australia website.
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Safety cabinets
Clean workstations, using biological safety cabinets or laminar flow clean air benches, are to be used for product and/or user protection. These types of equipment have different purposes and laboratory staff should be aware of the differences.
Laminar flow clean air benches
Laminar flow clean air benches protect the work (not the workers) from contamination and the air passes unfiltered onto the worker and into the laboratory. Laminar flow benches must never be used when handling pathogenic materials as any aerosols formed will be directed at the worker.
Biosafety cabinet
Virulent pathogenic organisms must be handled in a biosafety cabinet where contaminated air is passed through a high efficiency particulate air (HEPA) filter. There are three types of biological safety cabinet, Class I, Class II and Class III:
Class I: inward flow of air away from the operator and a HEPA filter is used before exhaust air is discharged from the cabinet
Class II: protects the operator by use of an air barrier and in addition a flow of filtered air passed over the work to prevent it becoming contaminated. A HEPA filter is used before exhaust air is discharged from the cabinet.
Class I and II cabinets are completely free-standing and must not be directly connected to ducting which has outside vents as wind may interfere with operator protection.
Class III: completely enclosed unit with built-in air locks for introducing and removing materials. Both incoming and outgoing air passes via HEPA filters. This class of safety cabinet is used for work with high-hazard micro-organisms.
Any procedure, such as using blenders, shakers or sonicators, that is likely to produce infectious aerosols should be carried out in a biosafety cabinet.
Details of the construction and performance requirements: Australian Standard AS2252 – 1985, Biological Safety Cabinets Parts 1,2, and 3. See the Standards Australia website.
All biological cabinets must be inspected and maintained by a registered repairer every 12 months.
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Decontamination
Disinfectants
Whenever possible, decontamination should be achieved by sterilisation in an autoclave (steam heat under pressure). Disinfectants should only be utilised where sterilisation is not possible, for example, in large spaces, surfaces and delicate instruments. Disinfectants should be chosen on their effectiveness to deal with the specific type of micro-organism.
The main uses for disinfectants are:
washing – such as, discarded containers, re-useable pipettes
wiping down benches and work surfaces at end of day
regular cleaning of equipment – such as water baths, incubators, centrifuges, freezers, refrigerators.
The following are some commonly used disinfectants:
ethyl or isopropyl alcohol, 80 per cent aqueous solution
chlorine as hypochlorite solution
iodine in aqueous or alcoholic solution (Formalin)
phenolic disinfectants – Lysol, Chloroxylenol.
Sterilisation
Steam heat autoclaves are used for sterilisation. Only properly trained staff should use the autoclave and care must be taken to ensure the load reaches the required temperature and remains at that temperature for the prescribed time.
Visual indicators such as Browne's tubes or heat-sensitive autoclave tape should be used routinely. Monthly checks of sterilising efficiency should be carried out by using spore strips, and times for sterilisation must be determined according to the load. Minimum sterilisation times after attainment of the required temperature are:
15 minutes at 121°C
2 minutes at 132°C.
The standard reference book on this topic is Introduction to Sterilisation and Disinfection by J Gardner and M Peel, Churchill Livingstone (1986).
Animals
Animals can be an important source of infection which may be acquired by man via ingestion, inhalation, eye contact, skin lesions or bites.
Other than the general safety procedures already given, the basic principle to be observed in procedures requiring the use of animals is that handlers should be properly trained and/or supervised to minimise the risk of accidental injury or infection.
Incorrect handling of an injured, frightened or nervous animal can easily result in injury to the handler or animal, and personnel should always be concerned not only with their own safety but also with the safety of animals in their care.
If you are uncertain about correct methods of restraint or handling of an animal, do not do so until you have obtained advice from trained personnel.
As a prophylactic measure all staff working with animals should be immunised against Tetanus.
Cages and racks should be demountable and autoclaveable, and when in use should be labelled to indicate the infectiousness of their contents.
All refuse and carcasses must be carefully disposed of in the correct manner, preferably by autoclaving and incineration. Animal houses should have sterilisation facilities in their own areas.
All animal users should be aware of the procedures governing the housing and care of animals in the University animal house areas. A copy of these procedures may be obtained from Animal Care Services on (+61 8) 6488 6606 or via: http://www.research.uwa.edu.au/staff/animals/animal-services .
Waste disposal
All infectious wastes should be disposed of in accordance with both federal and state regulations, and the following procedures should be followed:
All contaminated waste material shall be sterilised, preferably by autoclaving, before disposal, preferably by incineration.
Culture or fluids which may contain viable organisms shall not be poured into sinks or drains.
Solid contaminated materials shall not be placed in waste bins.
All samples, remains, disposable equipment, animal carcasses, tissue, fluids, faeces and bedding should be regarded as contaminated.
Aerosol cans or other sealed containers may explode if autoclaved or incinerated and must be surface sterilised only (using a suitable procedure).
Re-useable contaminated glassware should be disinfected or autoclaved or both before cleaning.
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Recombinant DNA techniques
Researchers who wish to carry out experiments which involve recombinant DNA techniques for the production of material incorporating recombinant DNA molecules unlikely to occur in nature must submit a proposal giving details of the project to the UWA Institutional Biosafety Committee (IBC) for approval.
The Gene Technology Act 2000 and Regulations have been produced to eliminate any possible hazard for occupational, public health and for the environment which may be associated with any genetic manipulation (GM) techniques. The safety of recombinant DNA work ultimately depends on the individuals conducting it.
The Biosafety Committee is available to provide advice to proponents about the Gene Technology Act 2000 and regulations. In particular it reviews applications for experiments as set out in the appropriate schedule of the regulations.
The committee serves a number of purposes such as:
maintaining surveillance over all known potentially biohazardous procedures and situations undertaken or existing with the University's sanction
ensuring that the highest safety standards and practices are observed
assessing all proposals to establish potentially biohazardous activities and for their approval or rejection – including all projects involving recombinant DNA techniques.
Currently the Committee and UWA Safety and Health play a broad role on behalf of UWA:
To apply for certification of laboratories of PC 2 standard before they are used for GM work.
To ensure that physical containment facilities and all containment standards continue to meet requirements.
To review applications for experiments as set out in the appropriate sections of the regulations.
To maintain a register of approved projects and associated experimenters involving Recombinant DNA work.
To advise the Office of the Gene Technology Regulator (OGTR) immediately of any accidents and unexplained illnesses or absences and subsequently provide a report on the investigation of such incidents.
To provide an annual report to OGTR.
To be responsible for ensuring that all personnel involved in Recombinant DNA work have sufficient training in microbiological techniques and the handling of pathogens. Training will be arranged through the Biosafety Committee where necessary.
To monitor the health of personnel working with Recombinant DNA.
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Levels of containment
There are three levels of physical containment for laboratory work with recombinant DNA. The classification is dependent upon the nature of the technique. The levels are referred to as PC 2, PC 3 and PC 4, with PC 4 being the highest level of containment.
Irrespective of the level recommended, there are certain practices that are minimum requirements for all levels.
PC 2 level
The laboratory must contain a hand basin with hot and cold water and a 'hands free' eye wash from which water flow remains constant (hand-held drench hoses and squeeze bottles are not suitable).
Laboratory doors must be closed when work is in progress.
Pipetting by mouth is prohibited.
Eating, drinking, storage of food and drinks, application of cosmetics and smoking is prohibited in the laboratory.
Laboratory coats or preferable back-fastening gowns must be worn in the laboratory and discarded on leaving.
Hands should be washed with soap and water before leaving the laboratory or whenever contaminated with cultures or culture extracts.
Laboratory should have access to an autoclave in the same building and all microbiological waste must be autoclaved prior to disposal.
Equipment used for handling cultures or contaminated material and which is not readily autoclaved must be disinfected with a suitable disinfectant after use and before disposal or being washed.
Work benches and surfaces should be decontaminated with an appropriate disinfectant solution after use.
All disinfectants must be made up daily.
All technical procedures must be performed in a way that minimises the creation of aerosols. In particular operations such as sonication or vortexing which may generate aerosols are to be carried out either:
in a biological safety cabinet when the material is in a large sealed container or in an open container
or
in a small sealed container in the laboratory.
Biological safety cabinets must be either Class I or Class II as specified in Australian Standard AS2252 – Parts 1 and 2. The cabinet must be able to be decontaminated with formaldehyde and the gas safety purged to atmosphere on completion.
Material taken out of the laboratory to a biosafety cabinet, autoclave or elsewhere should be carried in a closed, unbreakable outer container.
Laboratory doors must carry a sign clearly designating the level of containment and a similar sign should be posted in the laboratory giving procedures for the designated level of work.
Laboratory entrances and storage units (such as, refrigerators, freezers) in laboratories used for recombinant DNA work should be posted with the universal biohazard symbol.
Constant Temperature Rooms may be certified as PC2 facilities even if they do not contain sinks or coat hooks. Work shall otherwise follow the procedures required of PC 2 facilities.
PC 3 level
In addition to the requirements for PC 2 level:
Laboratories must not be situated adjacent to or open on to a corridor used by general public.
Walls, ceilings, floors, bench tops and finishes shall be smooth, impervious and easily cleanable.
Equipment shall be dedicated to the area.
All aerosol-producing equipment, such as that for sonication and vortexing, must be kept in the biosafety cabinet.
The biosafety cabinet must be located outside the influence zone of air inlet and exhaust registers, doorways and traffic ways.
The biosafety cabinet and/or laboratory must be decontaminated with formaldehyde gas after spills of contaminated material; also it must be possible to decontaminate the cabinet and room independently with formaldehyde and for the gas to be purged to atmosphere on completion.
Protective clothing should not be worn outside the laboratory and must be transported to the decontamination area in sealed bags or boxes.
Laboratories must be isolated from other areas by an airlock having two doors in series, each fitted with automatic closers.
Laboratories should be maintained at a minimum negative pressure of 50 Pa when both doors are closed and 25 Pa when one door is open. The negative pressure should be achieved by means of an independent room exhaust discharging to open air through a filter.
Replacement air should be drawn into the room via a filtered and adjustable aperture. Filters should have a minimum 95 per cent collection efficiency for all particles above five micrometres.
Hand basins should have foot- or elbow-operated taps and be located near the exit.
No other work should be done in the laboratory simultaneously with work requiring PC 3 containment.
No one may enter the laboratory for cleaning, servicing of equipment, repairs or other activities unless the principal investigator has been informed and the laboratory surfaces have been disinfected.
A program of pest control for insects, rodents and other pests shall be instituted.
PC 4 level
The requirements for a PC 4 laboratory are much more stringent than for other containment facilities, and the following are the requirements in addition to those for PC 2 and PC 3 levels.
Laboratories and airlocks must be ventilated independently of the rest of building by an air supply and exhaust system giving a continuous minimum negative pressure of 50 Pa in the laboratory and 25 Pa in the airlock.
Supply and exhaust air must be filtered by HEPA filters.
A double-ended autoclave should be fitted and sealed in the wall of the laboratory and used to sterilise all material being removed from the laboratory.
Waste water should be drained to a system where suitable treatment to sterilise or decontaminate it can take place.
No other work should be done in the laboratory simultaneously with work requiring PC 4 containment and only authorised personnel are to enter the laboratory during PC 4 work.
