All occupations have their special hazards but although there are risks of infection in laboratory work with microorganisms, they are well recognized and can be contained by good laboratory practice. That this is so is shown by the very small number of infections acquired in recent years in clinical laboratories in relation to the large number of staff employed.
Routes of Infection
Inoculation
Literally, inoculation means the introduction of material into an ‘eye’ as in horticultural practice of grafting, and by analogy it covers the deliberate or accidental introduction of infection into the body, particularly by a breach of its surface. Thus, it covers the introduction of infection into the eye by splashing or by rubbing with contaminated fingers, injection through the skin by needle-stick injury or the bite of an ectoparasite, incision with a sharp instrument or broken glass, and injunction by the rubbing of material on to the skin or a mucous membrane. There are a few pathogens that can penetrate the skin or mucosa spontaneously, but usually the epithelia have small breaches of continuity that allow a wider range of microbes to reach the tissues.
Ingestion
Infection by the oral route may take place by the licking, sucking or accidental swallowing of infective material. It is especially liable to occur in the mouth-pipetting of cultures or other infected fluids. The cotton-wool plug in the upper end of a pipette does not protect against the risk and the finger placed at the upper end to control the pipette also touches the lips and may itself to contaminated. Infection may also be ingested after touching the mouth with contaminated fingers or writing instruments, or by licking labels contaminated by the fingers, or during eating, drinking or smoking in the laboratory.
Inhalation
Infection may take place by the breathing-in of an infected aerosol or dust. An aerosol is a cloud of small droplets of liquid in air. It usually contains many droplets smaller than 0.1 mm in diameter and these droplets dry rapidly to become solid residues, called ‘droplet nuclei’ so small, e.g. 1-20 um diameter, that they may remain air-borne for up to several hours.
Clouds of infective dust or spores can be released by the opening of containers of freeze-dried cultures or the withdrawal of cotton-wool plugs that have dried after being wetted by culture fluid. Convectional and other air currents can disperse aerosols and dusts widely within a laboratory and also into adjacent rooms. Particles larger than 5 um in diameter are mainly deposited in the nose and throat, whilst smaller particles can reach the bronchi and lungs.
Hazardous procedures
A number of procedures and situations afford a particular risk of infection. They include the following procedures:
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Use of syringe and needle. The operator may puncture his skin with the needle during use or disassembly of the syringe, or another person may accidentally puncture himself with an improperly disposed, used needle.
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Pipetting. Infective material may be ingested in mouth-pipetting or be disseminated on to the surroundings in drips or aerosol, especially if the last drop is blown out.
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Inoculating loop. Vibration of an inoculating wire or ‘loop’, especially if more than 4-5 cm long may cause splashing and aerosol production.
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Petri dishes. Water of condensation on the agar or in the lid may become contaminated and be split on the fingers or the bench.
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Mixing, shaking or homogenizing. Shaking produces an aerosol even in a closed container, which can be released on opening the container within a period of several minutes, or through an imperfect seal.
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Centrifugation. Vibration can generate aerosol within the container and this may be released during the operation or on opening.
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Freeze dying. Some contamination of air currents from the material being dried is inevitable and the process should be carried out in an otherwise unoccupied room. The opening of the sealed ampoules is also hazardous.
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Stoppering tubes. If a tight stopper is applied forcibly or if the tube is faulty, the glass may break and cut the hand, and contents may be spilled.
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Safety cabinets. Unless properly installed, maintained and used, these cabinets can disseminate infection into the air of the room or the exhaust duct.
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Automated apparatus. It can become contaminated and also splash samples on surrounding surfaces.
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Animal procedures. Work with animals, including inoculation, collection of samples and performance of necropsy, affords many opportunities for injury and infection.
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Transport of specimens. Improperly packaged and inadequately closed samples may leak and contaminate wrappings and attached forms during delivery to the laboratory.
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Disposal. Contamination, injury or infection may occur at many points in the procedures for collecting discarded cultures, specimen containers and used equipment, and their documentation, washing or incineration. Discarded ‘sharps’ (needles, scalpels, etc.) are a particular danger.
Safety Organisation
Safety codes
Increasing realization of the importance of laboratory infection has led to the establishment of regulatory authorities in a number of countries. A further important influence in Britain has been the passing of Health and Safety at Work Act 1974 which gives legislative grounds on which current codes of practice and their enforcement are based.
Two principal documents provide guidance on the prevention of laboratory infection and containment of pathogens in Britain. These are (1) the code of practice for the prevention of infection in clinical laboratories and post-mortem rooms – the ‘Howie Code’ (1978) which considers infection risks in all types of clinical laboratory, and (2) the Report of the Advisory Committee on Dangerous Pathogens on the Categorisation of pathogens according to Hazard and Categories of Containment (ACDP) 1984. Laboratory workers in Britain must be familiar with both documents.
In Britain, pathogens (bacteria, chlamydias, rickettsias, mycoplasmas, viruses, fungi and parasites) are categorized into four hazard groups, based on the inherent hazard of the organism.
Group 1
An organism that is most unlikely to cause human disease.
Group 2
An organism that may cause human disease and which might be a hazard to laboratory workers but is unlikely to spread in the community. Laboratory exposure rarely produces infection and effective prophylaxis or treatments are usually available.
Group 3
An organism that may cause severe human disease and present a serious hazard to laboratory workers. It may present a risk of spread in the community but there is usually effective prophylaxis or treatment available.
Group 4
An organism that causes severe human disease and is a serious hazard to laboratory workers. It may present a high risk of spread in the community and there is usually no effective prophylaxis or treatment.
Only pathogens are listed in the ACDP report. Most fall into group 2, but those falling into groups 3 and 4 are listed in the table 15.1 below.
An important feature of the ACDP Report is the categorization is the guide to the containment necessary when working with an organism. However, in circumstances where the nature of the work or volume of materials used increases the hazard, the categorization may be raised to a higher level.
Conversely, if the risk is reduced by working, for example, with a non-virulent organism or a variant that cannot survive except in special cultural conditions, the category may be lowered provided a proper risk assessment has been made and agreed with those working with the organism, and perhaps also with the Health and Safety Inspectorate.
Containment Level 1
The categories, or levels, of laboratory containment are numbered to accord with the category of hazard an organism presents. Since organisms falling into hazard category 1 (i.e. group 1) are normally harmless, the requirements for work with these organisms are only that the laboratory can be cleaned easily and must have washing facilities.
Containment Level 2
Most pathogens dealt with in clinical laboratories fall into category 2 and will therefore be handled in containment level 2. The laboratory should be easy to clean, access should be limited to laboratory personnel and other specified persons and it should be of adequate size (24 m3 per worker). Laboratory coats preferably back or side fastening must be worn and an autoclave for the sterilization of waste must be readily accessible. Hands and benches must be disinfected after work and material for disposal must be stored and handled safely. All clinical laboratory suites must contain a class 1 microbiological safety cabinet.
Containment Level 3
This category requires in addition that the laboratory should be sited in an area away from general circulation and has a biohazard sign at the entry. A continuous airflow into the laboratory must be maintained during work with pathogens and must be exhausted via a HEPA (high efficiency particle air) filter and procedures must be conducted in a class 1 or 3 safety cabinet. Gloves must be worn for all work with effective materials.
Containment Level 4
This is the strictest category of containment. It requires sophisticated air movement control and filtration as well as the use of sealed safety cabinets of class 3, a double-ended autoclave and many other safety features. Very few laboratories should do work with organisms presenting the highest level of risk and so need such expensive facilities.
Postal regulations
These are special regulations for sending infectious material by post to ensure that the containers do not get broken and their infected contents cannot leak. These are also strict special regulations relating to the transport of group 4 pathogens.
Reception of specimens
Specimens must be received in a separate area in the laboratory which must not be part of an office or in a public corridor. Where there has been leakage of a specimen either due to a loose container cap or due to breakage of the container, a decision must be taken by a senior member of staff as to whether the specimen is to be discarded or whether the difficulty of obtaining a further specimen, e.g. cerebrospinal fluid, is such that the leaking container should be taken to a safety cabinet for transfer of the specimen to a fresh container. Cover any potentially infected material spilt at the reception area with a cloth or paper towel soaked in disinfectant and leave for at least 10 minutes before mopping up with cloths, or cleaning up with a brush and pan, and placing in an infected-waste container.
Procedures in the laboratory
For most clinical specimens the appropriate containment in the laboratory is at level 2. But where the specimen is known, or likely, to contain a category 3 pathogen, e.g. sputum, faeces from a known case of typhoid, it is at level 3. The basic rules of good laboratory discipline apply at all times so that the worker may avoid infection of himself and others. Any action likely to result in the generation of infective aerosol or the splashing of infective material must be carried out in a safety cabinet.
Mishaps with infective material
Encourage cuts or puncture wounds to bleed and then wash with soap and water. If the eye is splashed rinse at once either with tap water or with irrigating solution held in the laboratory first aid kit.
Discard jars
At the start of each day, empty contents from the previous day, carefully clean the jar; preferably disinfect it by heating at least 65 oC for 10 minutes and refill with fresh disinfectant diluted accurately to the correct concentration, e.g. hypochlorite to give 1000 or 10 000 p.p.m. of available chlorine or phenolic disinfectant, e.g. hycolin, at 1% or 2% concentration.
Discard material
This must be disposed of safely. Send non-infected material for incineration where appropriate (e.g. paper and plastics). Put broken glass in robust containers to protect workers from injury.
Pipetting
Use a rubber teat or automatic suction device, never the mouth. Take care neither to draw fluid up as far as the top of the pipette nor to draw bubbles through the liquid. When transferring fluid to another container, first place the tip of the pipette well inside the mouth of the receptacle in contact with its wall and then allow the contents to run gently down the wall.
Hypodermic syringes
Sharp instruments and needles should be avoided as far as possible in Microbiology laboratories. Use plastic disposable, not glass syringes. Ensure the needle is firmly attached.
Centrifuging
Use only centrifuges with sealable safety buckets. Use the centrifuge strictly according to the operating instructions which should be posted beside it. to avoid breakages by vibration or unseating of buckets, balance the loads accurately and symmetrically and take care to fit the buckets and trunnions properly in place.
Immunization
Staff of laboratories dealing with infective materials should be immunized against diphtheria, tetanus, poliomyelitis, tuberculosis, typhoid, rubella and hepatitis B. Where immunization was not given in childhood, a full course should be given. Recognized prophylactics should also be given where special pathogens, e.g. anthrax, plague, rickettsia, coxiella, Clostridium botulinum, are worked with.
Safety Cabinets
These cabinets provide a barrier between the worker and infectious material and are designed to prevent infection by splashing or by aerosol.
Disinfectants
Disinfectants are needed in the Microbiology laboratories for the skin, work surfaces, discard jars and spillages. Use as few kinds as possible to avoid confusion and explain their function and limitations to all who use them.
Skin and work surfaces can be decontaminated with 70% ethyl alcohol (industrial methylated spirits with 1% glycerol as an emollient for skin use). This will kill vegetative bacteria and some viruses. If there is any fire hazard and especially if viruses or bacterial spores pose a problem, sodium hypochlorite solution, a cheap disinfectant and rapidly lethal to most bacteria and viruses, may be used.
However, it is ineffective against Mycobacterium tuberculosis, is neutralized by organic material and is corrosive to metals, but it is free from any marked or persistent irritant or toxic effect. For disinfecting clean surfaces or skin a 1% solution of commercially available hypochlorite solutions such as Chloros or Domestos is adequate. These proprietary disinfectants are 10% solutions of sodium hypochlorite containing 100 000 p.p.m. of available chlorine. Prolonged exposure of skin to hypochlorite should be avoided, the disinfectant being washed off as soon as possible under running water.
Each work place in the laboratory should be provided with one or more deep plastic discard jars filled with disinfectant for the disposal of contaminated pipettes, slides, ineffective residues, etc. a bottle of disinfectant must be kept for spillages. For discard jars, spillages or any situation where there is organic or tuberculous material a phenolic disinfectant should be used as these disinfectants are more resistant to inactivation than hypochlorine.
If work with viruses being is being carried out, pipette jars should be filled with hypochlorite solution containing 2500 p.p.m. available chlorine and for blood spillage 10 000 p.p.m. may be used. If there is much organic matter in a virus spillage or if the corrosive effect of hypochlorite might affect equipment, 2% glutaraldehyde should be used. Constant exposure to glutaraldehyde can give rise to sensitization.
