School of Physics Safety Manual

12. BIOLOGICAL HAZARDS

General

It is imperative that all personnel who deal with biological experimental procedures should be conscious of the possible hazards that may arise. If in doubt you must consult your veterinary or medical/clinical partners.

A bacteriological risk arises from any procedure which releases micro-organisms into the environment or which otherwise allows them access to the human body. Infection may be initiated by inhalation, ingestion, through the broken or unbroken skin, or the conjunctiva.

Many laboratory activities with fluids produce aerosols. When a fluid surface is broken large number of small droplets are produced. This may happen when a bubble bursts, when two solid surfaces separated by a film of liquid are parted, when one liquid is poured into another and when drops of liquid are allowed to fall on to a solid surface. The larger droplets fall to the ground, but the smaller ones evaporate rapidly and if the fluid contains bacteria these remain suspended as infected air-borne particles or droplet nuclei. The small particles, between 1 and 5 μm remain suspended for long periods and if inhale penetrate directly into the lung. Larger particles are removed in the upper air passages.

Aerosol production is dangerous not only to the operator but to all other persons in the vicinity. Even screw-capped bottles may present some hazards. A thin film of liquid may be present between the rim of the bottle and the rubber liner. When this film is broken by removing the cap aerosols are formed.

Gross contamination of surfaces and of clothing by spilled or broken cultures may result in infection by the hand-to-mouth route, to infection of existing skin lesions and to eye infections. The possibility of systematic infection by the conjunctival route must also be considered.

Direct ingestion is usually associated with mouth pipetting, failure to wash the hands, direct consumption of foods in the laboratory and smoking at the work bench.

Faulty techniques with hypodermic needles, and accidents with broken glass may lead to direct inoculation into the blood stream. Contamination of the skin with blood may lead to infection directly or through minor abrasions, e.g. Hepatitis B.

The ideal specimen container for pathological material must be easy and convenient to use by the patient and doctor without the outside being contaminated. It must be leak proof and virtually unbreakable: it should not create undue aerosol or spillage hazards when opened in the laboratory, and it should be easily sterilised, washed, or disposed of.

Screw-capped bottles may present some hazards (see Section 8.2.5). This risk is very much greater in containers that have ‘snap-on’ or ‘pop-up’ closures or bungs, or have ‘screw-in’ plugs. A larger film of liquid is easily trapped between the top of the container and the closure particularly after shaking in transit. It is very difficult to open these containers gently, as is possible with a screw-capped bottle; and the abrupt movement releases large amounts of aerosol and often a visible spray. These containers and many others in which any part of the closure is inside the tube may be dangerous and should not be used. Unfortunately they have become very popular for the collection of blood specimens for haematological and biochemical purposes. Hitherto these have been regarded as harmless, but a proportion of these samples is now known to contain Australia-Antigen.

Some screw-caps, particularly those made of plastic, seem to become loose spontaneously.

Sera sent from one laboratory to another should be dispatched in the smaller glass screw-capped (Bijou) bottles.

Good Working Practices

• All procedures should be performed so as to minimise the production of aerosols.
• The laboratory door should be closed when work is in progress.
• Laboratory coats must be worn in the laboratory and removed when leaving the laboratory.
• Personal protective equipment, including protective clothing, must be:
• stored in a well-defined place;
• checked and cleaned at suitable intervals;
• when discovered to be defective, repaired or replaced before further use.
• Personal protective equipment which may be contaminated by biological agents must be:
• removed on leaving the working area;
• kept apart from uncontaminated clothing;
• decontaminated and cleaned or, if necessary, destroyed.
• Eating, chewing, drinking, taking medication, smoking, storing food and applying cosmetics is prohibited in laboratories.
• Persons working in biological laboratories should adhere to a strict code of personal hygiene
• Hands must be decontaminated immediately when contamination is suspected, after handling infective materials and before leaving the laboratory. When gloves are worn, these should be washed or preferably changed before handling items likely to be touched by others not wearing gloves, for example, telephones, paperwork, computer keyboards and, where practicable, equipment controls should be protected by a removable flexible cover that can be disinfected.
• Mouth pipetting is forbidden.
• Bench tops and work surfaces should be regularly decontaminated according to the pattern of work and should be cleaned after use.
• Used glassware and other materials awaiting disinfection should be stored in a safe manner. Pipettes, for example, if placed in disinfectant, should be totally immersed.
• Material for autoclaving should be transported to the autoclave in robust containers without spillage.
• There must be safe storage of micro-organisms.
• All waste material, if not to be incinerated, should be disposed of safely by other appropriate means.
• Accidents and incidents must be immediately reported to and recorded by the person responsible for the work or other delegated person.
• Effective disinfectants should be available for immediate use in the event of spillage.
• The laboratory should be easy to clean. Bench surfaces should be impervious to water and resistant to acids, alkalis, solvents and disinfectants..
• Minimum paper only should be in laboratories ie essential paperwork and laboratory books only.

• All infectious and toxic materials should be correctly labelled and storage areas should show appropriate warning notices.
• Laboratories handing biological materials which could cause illness should have a system whereby illness is reported without delay.
• Great care should be exercised in using hypodermic syringes. Apart from the risk of accidental self inoculations, spraying may occur if a needle becomes loosened from the syringe during an injection.
• Laboratory personnel must receive suitable and sufficient information, instruction and training in the procedures to be conducted in the laboratory.
• Training received must be recorded on a laboratory proforma and signed by the trainee. This should also include details of relevant documents read.
• Every researcher must have a nominated academic supervisor responsible for ensuring compliance with these rules.
• Individuals working with micro-organisms must ensure that other persons eg cleaners, maintenance personnel, visitors etc are not exposed to biological hazards.
• Portering staff must only deal with general waste and do not handle autoclaved, biohazardous or other similar waste. This must be dealt with (including transportation to the wheelie bins) by lab workers.
• Project students and other undergraduates working with micro-organisms require close supervision, the safety and security of these individuals is paramount

Handwashing Facilities

Hand basins with hot and cold running water must be provided in all laboratory rooms. There should be at least one basin in each room. In rooms where a large number of people work, e.g. more than 10, it is desirable that two basins be provided. The basins should be near to the doors. Ideally, taps should be operable without needing to use hands.

Automatic liquid soap dispensers should be avoided in favour of tablets of toilet soap regardless of the alleged economy of the former. Many of these dispensers must be operated by dirty or contaminated hands before they yield soap. It may not be obvious when they are empty.

Paper towels in dispensers are much more satisfactory than Turkish or huckaback towelling. These cloth towels are usually changed on a rigid time basis and may remain wet and dirty for long periods. Roller towels are acceptable only if they are of the continuous flow type and are properly maintained and promptly replaced.

As a general rule hands should be washed, preferably with a bactericidal soap, after completing any work and always before leaving the laboratory.

Health

Individuals with medical conditions which predispose them to infection (eg eczema, compromised immune systems, diabetes etc) are at a special risk. Everyone must notify their supervisor of any illness or other medical condition that may compromise the immune system and may make them more susceptible to hazards, which may arise through working with micro-organisms.

Health surveillance is required under COSHH where:

• there is an identifiable disease which may be related to workplace exposure;
• there is a reasonable likelihood that exposure may happen;
• there are valid techniques for detecting indications of the disease or its effects.

Autoclaves

Most biological agents (including bacterial and fungal spores) can be rendered non-viable by exposure to steam under pressure. The hazard from adequately treated material should be very low. Sterilisation depends on steam penetrating to all parts of the load. Lids must be removed and plastic bags undone. The efficiency of sterilisation should be verified by one of the following methods:

• A thermocouple placed in the load will indicate how long the load takes to heat up to the chosen sterilisation temperature and how long the autoclave should be maintained at this temperature. A safety margin should be included to ensure sterilisation.
• Spore strips may be placed in the load but, as they have to be incubated, do not give an instant indication.
• Some indicator devices (eg “Thermolog” strips) may give a reasonably reliable indication of sterilisation and (subject to risk assessment) be suitable for day to day use where thermocouple tests are impracticable.
• Ordinary autoclave tape, however, does not give a reliable indication of sterilisation.

Autoclave procedures

• Waste destruction conditions need to be verified and specified to fall in line with appropriate licences for the area. The norm is 1 hour at 121ºC (15psi).
• Procedures for final disposal need to be specified in the procedure. Portering/cleaning staff must not be required to handle such material.
• Autoclaves must be operated by trained, competent staff and manufacturer’s instructions must be followed.
• Heat resistant gloves and a face visor should be worn.
• Loads containing bottled fluids must be cooled to below 80ºC before removal from the autoclave (otherwise they may explode).
• Items which may explode, release toxic fumes etc or corrode surfaces must not be autoclaved.
• Autoclaves must be periodically inspected (as pressure vessels), tested, maintained and records must be kept (Pressure Systems Safety Regulations 2000).

Disinfection

Disinfection is a less reliable means of sterilising materials than autoclaving. Disinfectants must be chosen carefully as there is no universal disinfectant, all have disadvantages. Disinfectants may deteriorate on standing or be inactivated by detergents, organic matter etc. and most are toxic or irritant. Some common types are listed below:

Hypochlorite solutions:

• Commonly recommended concentrations – 1,000 ppm for surface decontamination, 2,500 ppm for discard containers, 10,000 ppm for spillages.
• These are active against bacteria (including spores) and viruses but have limited activity against fungi and tubercule bacilli.
• Are compatible with anionic/non-ionic detergents but corrode many metals and damage rubber and are inactivated by organic materials and so need frequent changing.

Chlorine releasing granules:

• Usually contain sodium dichloroisocyanurate (NaDCC) and may also contain absorbent powders. They have a relatively long shelf life and are useful for spillages.

Clear soluble phenolics:

• Active against vegetative bacteria (including tubercule bacilli) but not active against spores and have a limited effect on fungi. They are not active against many viruses (particularly if not lipid containing).
• Compatible with anionic/non-ionic detergents and metals and inactivated by rubber and some plastics
• Hycolin has a new formulation and is not reliable.

Glutaraldehyde:

• Has similar range of activity to hypochlorites but should not corrode metal.
• Does not readily penetrate organic matter and is relatively unstable once inactivated.
• Is a potent allergic sensitizer.

Alcohols:

• Are normally used as 60-80% vv solutions in water.
• Are active against protozoa and many viruses and vegetative bacteria (but not tubercule bacilli).
• Can be used as a disinfectant skin rub (often with addition of 5% wv chlorhexidine)
• Do not readily penetrate organic matter.
• Are flammable.

Virkon:

• Claimed to be active against many types of organism.
• Relatively non-toxic and non-corrosive.

Disposal of Biological Waste Material

It is important that biological waste is disposed of safely. Appropriate sterilisation procedures must be used prior to disposal of any material contaminated with micro-organisms.

Some guidelines:

• Unlicensed Waste for autoclaving. Material should be placed in clear plastic autoclave bags for standard autoclaving at 121°C for 60 minutes before disposal.
• Licensed Waste eg plant pathogens. Material should be placed in RED plastic bags for autoclaving at 121°C for the time specified on the licence (usually 1-2 hours).

Material from the microbiology suite should then be placed in a yellow bag and taken to the yellow waste for incineration wheelie bin in the stores yard. Other autoclaved material such as large quantities of soils should be placed in a black bag and placed in the general waste wheelie bin.

• Unlicensed waste for incineration. Material that is unsuitable for autoclaving (including small quantities of some chemical waste that requires incineration) would be placed in special YELLOW Biohazard labelled bags. These bags should be filled no more than ¾ full and securely sealed. The bag must be labelled with the lab., building and date of filling. This bag should then be placed in the yellow wheelie bin in the stores yard. The key is with the storekeeper. The material is then sent for incineration. Keep this waste to a minimum.

Further guidance on standard operation procedures for waste disposal is given in Appendix 5 of The Management, Design and Operations of Microbiological Containment Laboratories.

Action in the event of spillage

 

There must be contingency plans for dealing with foreseeable emergencies. These could include spillage control, room evacuation, fumigation and decontamination and, if there is a risk of infection - first-aid and medical treatment (prophylazis) and health surveillance and counselling of exposed people.

Items required for dealing with spillages etc must be readily available and all workers must know the procedures. Guidance is given in Appendix 3 of The Management, Design and Operation of Microbiological Containment Laboratories.

 

In the event of an accident/incident/emergency, immediate steps must be taken to mitigate the consequences. Only people essential for carrying out repairs and other essential work may be permitted in the affected area and they must be provided with appropriate personal protective equipment and any necessary equipment or plant. Employees (or their representatives) must be informed as soon as practicable after an incident or accident that has (or may have) resulted in the release of a biological agent that could cause severe human disease. Employees must report such incidents or accidents forthwith.

If there is a risk of airborne infection the laboratory must be evacuated as quickly as possible. It may be necessary to fumigate the room before reoccupation.

• Spillages should be contained and covered with disinfectant granules or absorbent paper/cloth soaked in disinfectant.
• The disinfectant should be allowed to act for at least 15 minutes.
• The debris should be swept gently into a dustpan using a piece of board or stiff card.
• Any residual pieces of glass etc should be picked up with forceps or swabs.
• Debris should be put in a suitable container for disposal by a safe route; and
• Further disinfectant should be applied to contaminated surfaces.
• Rooms must not be re-occupied until it is safe to do so.

People attending casualties should avoid becoming contaminated themselves.

• Contaminated clothing must be removed as quickly as possible.
• Remove contamination of skin/eyes/mouth by thorough washing with clean water.
• Eyes should not be rubbed nor skin scrubbed.
• Small puncture wounds should be encouraged to bleed; minor cuts and similar lesions should be washed with soap and water or a suitable detergent before being thoroughly washed and dressed; and
• Medical advice must be sought if there is a risk of infection.

Surveillance of exposed persons and possibly their close contacts may be required. Counselling may be necessary. If there is a health risk to the community there must also be contingency plans, made in consultation with local community physicians to limit spread:

• Identification and tracing of contacts;
• Testing, vaccination or prophylaxis of contacts;
• Isolation and treatment of infected people;
• Control of animal vectors

Containment measures

The term “containment” describes the way in which biological agents are managed in the laboratory environment so as to prevent or control the exposure of laboratory workers, other persons and the outside environment to the agent(s) in question. This is achieved by a combination of measures.

Primary containment, ie, the protection of the worker and the immediate environment can be achieved through a combination of good microbiological practices/ techniques and the use of appropriate safety equipment, eg, microbiological safety cabinets. Further protection may be achieved through the use of appropriate immunisations, although this should be seen only as a useful supplement to reinforce procedural controls and the use of safety equipment, not the sole protective measure.

Secondary containment, ie, the protection of the people and the environment outside the laboratory can be achieved by a combination of laboratory design, engineering controls and operating procedures.

Containment measures must be reviewed at suitable, regular intervals and immediately if there is reason to suspect that the measures are no longer adequate or, if in the light of new scientific or technical knowledge, the assessment is inadequate. Laboratory containment measures must reflect the nature and severity of the biological hazard; an outline guide is provided in the table below.

Laboratory Containment Level

Containment Measures	1	2	3
Laboratory suite: isolation	Not required	Not required	Required
Laboratory sealable for fumigation	Not required	Normally required, depending on the workplace risk assessment	Required
Equipment
Surfaces impervious to water and resistant to acids, alkalis, solvents, disinfectants, decontamination agents and easy to clean	Required for bench	Required for bench and floor	Required for bench, floor, ceiling and walls
Entry to lab via airlock	Not required	Not required	Required
An inward airflow into the laboratory (negative pressure) must be maintained by extracting room air to atmosphere	Not required	Required where indicated in risk assessment	Required
Extract and input air from the laboratory must be HEPA filtered (H14 standard tested to 99.997% efficiency)	Not required	Not required	Single HEPA filters required for extract air, single HEPA filters for input air
Use of a microbiological safety cabinet/enclosure	Not required	Required where there is a risk of aerosol generation	Required, Class I/III cabinet or isolator
Autoclave	Required on site	Required in the building	Autoclave required in the laboratory
System of Work
Access restricted to authorised personnel	Required	Required	Required (key pad lock required)
Specific measures to control aerosol dissemination	Not required	Required so as to minimise	Required so as to prevent escape from primary containment
Shower	Not required (unless required for chemical safety)	Not required (unless required for chemical safety or if large volumes/high concentrations to be used)	Required – emergency use
Protective clothing	Suitable protective clothing required	Suitable protective clothing required	Suitable protective clothing required; footwear required where and to the extent the risk assessment shows it is required
Use of disposable gloves	Required where indicated in risk assessment	Required where indicated in risk assessment	Required
Efficient control of disease vectors	Required where and to the extent the risk assessment shows it is required	Required	Required
Specified disinfection procedures in place	Required	Required	Required
Biohazard sign displayed on laboratory door, fridges, freezers and transport containers	Required	Required	Required
Waste
Inactivation of biohazards in effluent from hand-washing sinks and showers and similar effluents	Not required	Not required	Required where and to the extent the risk assessment shows it is required
Inactivation of biohazards in contaminated material and waste	Required by validated means, where and to the extent the risk assessment shows it is required	Required by validated means	Required by validated means
Other Measures
Laboratory to contain its own equipment	Not required	Not required	Required, so far as is reasonably practicable
An observation window or alternative is to be present so that occupants can be seen	Required where and to the extent the risk assessment shows it is required	Required where and to the extent the risk assessment shows it is required	Required
Safe storage of biohazardous material	Required where and to the extent the risk assessment shows it is required	Required	Secure storage required
Telephone or intercommunication system between the laboratory and the clean outside area	Required	Required	Required
Written records of staff training	Required	Required	Required
Space for laboratory workers	There must be adequate space (at least 11m³) in the laboratory for each worker	There must be adequate space (at least 11m³) in the laboratory for each worker	There must be adequate space (at least 24m³) in the laboratory for each worker

Microbiological safety cabinets

Where used, be aware that there is a legal requirement to check these cabinets for operation and containment. High Efficiency Particulate Air (HEPA) filters used in microbiological safety cabinets are to be checked by a professional engineer twice per year, and the potassium iodide test is performed annually. Class 1 Safety Cabinets minimize escape of aerosols by drawing air in through the open front. The air passes through a high efficiency particulate air (HEPA) filter and is then discharged (usually to the outside). These cabinets protect the worker but not the work.

There is a device (anti-blow back valve) to prevent back flow of air if the fan fails. Class 2 Safety Cabinets are designed such that air is drawn through the open front of the cabinet, but instead of going upwards, it is drawn downwards through grills in the base of the cabinet. The air passes up the back of the cabinet and is HEPA filtered. A proportion is vented (again normally to the outside) while the rest is re-circulated downwards through the working area providing a curtain of filtered air. These cabinets protect both the worker and the work from contamination and are particularly useful for handling cell cultures. Class 3 and Class 1/3 Safety Cabinets provide a totally enclosed working area and the operator wears long rubber gloves which are attached to the front panel. Incoming air is HEPA filtered once and outgoing air twice. Both work and worker are protected but this type of cabinet is normally only necessary in Containment Level 3/4 laboratories. Laminar Flow Cabinets may be used for preparing media and pouring plates only. They can be horizontal or vertical flow type. With these cabinets, the direction of flow is outwards towards the worker providing a sterile working environment for procedures such as plate pouring only. Because of the outward flow of air there is no protection for the worker and therefore these cabinets must not be used for handling any pathological materials or cultures of any micro-organisms.

Animals

Any animals should be kept in suitable housing to which only authorised persons have access. Any laboratory animal, whether stock or experimental, may excrete an infectious agent in its faeces, urine, saliva or expired air. Infection of handlers is also possible by direct transmission during handling; from bites and scratches, from contact with or inhalation of dust from cages and bedding; and from contact with blood or with tissues removed at autopsy.

Infected animals may not be obviously ill; infections may be subclinical. Therefore all laboratory animals at all stages of their progress through an animal house must be treated as potentially infectious. Anyone bitten by an animal should be treated by a medical officer.

Reference material

• Health and Safety at Work Act (HASWA) 1974 and the European Communities Act 1972 and regulations within, including Control of Substances Hazardous to Health (COSHH) 2002 Regulations (as amended)
• The Management, Design and Operation of Microbiological Containment Laboratories. Advisory Committee on Dangerous Pathogens. HSE Books 2001
• Biological agents: managing the risks in laboratories and healthcare premises. Advisory Committee on Dangerous Pathogens. HSE Books 2005.

http://www.hse.gov.uk/biosafety/biologagents.pdf

• Categorisation of biological agents according to hazard and categories of containment. Advisory Committee on Dangerous Pathogens. 1995. Fourth Edition
• The Approved List of Biological Agents. Advisory Committee on Dangerous Pathogens. HSE 2004. http://www.hse.gov.uk/pubns/misc208.pdf
• Work with agents listed by DEFRA as specified animal pathogens may require a DEFRA licence(s). Advice should be sought from the Biological Safety Advisor. The list of specified animal pathogens can be found at: http://www.defra,gov.uk/animalh/diseases/pathogens/index.htm
• Home Office Anti-Terrorism, Crime and Security Act (ATCSA) 2001 (http://www.homeoffice.gov.uk/security/terrorism-and-the-law/anti-terrorism-crime-security-ac/) and pathogens listed under Schedule 5 (http://www.opsi.gov.uk/acts/acts2001/ukpga_20010024_en_18#sch5)
• Safe Working and the Prevention of Infection in Clinical Laboratories (HSAC)
• Laboratory-Acquired Infections (Collins)
• Biological Safety: Principles and Practices (ASM).
• Air Transport - ICAO Technical Instructions for the Safe Transport of Dangerous Goods by Air. The Air Navigation Order 2005 SI 1970 provides the legal basis for the Air Navigation (Dangerous Goods) Regulations 2002 (SI 2786) (As amended)

• Road and Rail Transport – ADR European Agreement concerning the International Carriage of Dangerous Goods by Road

RID Regulations concerning the International Carriage of Dangerous Goods by Rail

The Carriage of Dangerous Goods and Use of Transportable Pressure Equipment Regulations 2004 (SI 568) as amended

• Maritime Transport - IMDG International Maritime Dangerous Goods (Code)

The Merchant Shipping (Dangerous Goods and Marine Pollutant) Regulations 1997 (SI 2367)