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Decontamination

 

Contaminated laboratory waste

Laboratory waste should be separated into colour-coded containers. The recommended colours are;-

Yellow - for incineration

Light blue or transparent with blue inscription - for autoclaving (but may be incinerated subsequently

Black - normal household waste: local authority refuse collection

White or clear plastic- soiled linen

There are three practical methods of treating contaminated laboratory waste: sterilization by autoclaving, chemical disinfection, and incineration. The first two are laboratory processes; incineration involves the transport of material off-site.  Sterilization and disinfection are not synonymous: sterilization implies the killing of all microorganisms; disinfection kills most microorganisms but depend greatly on the chemical used.

Autoclaves

Autoclaving involves the timed exposure of materials to steam above atmospheric pressure and hence at temperatures above 100oC. Autoclaves operate at high pressures and temperatures and their manufacture, installation, and use are regulated. In the UK, they are subjected to the Pressure Systems and Transportable Gas Containers Regulations 1989, which requires regular inspection and maintenance and regular checks on the effectiveness of the sterilization cycles. For most purposes, sterilization is achieved by exposure of material to 121oC for 15-20 min. Higher temperatures such as 126oC for 10 minutes, or 134oC for 3 minutes are permitted. Temperatures are monitored by thermocouples in modern autoclaves. Chemical and biological indicators are also widely used, the most well-known of which is the "autoclave" tape. Biological indicators is common use consist of strips or discs of filter paper which have been soaked in suspensions of Bacillus stearothermophilus spores, then dried and packed in porous envelopes or sachets. These are then placed in various positions in the load and after processing, the strips and discs are removed from their packing and placed in nutrient broth which is incubated for 24-48 hours. The presence of turbidity indicates failure to sterilize.

It was customary to place material for autoclaving In wire baskets and cylindrical buckets. While these containers are perfectly acceptable for bottled fluids, they are not suitable for potentially infected material since they do not contain spilled material. Therefore, shallow buckets or other "solid-bottomed containers" should be used for sterilizing infected material which may leak. It is important to ensure that the container used is not overlarge since it may lead to inadequate sterilization. Autoclavable plastic bags are now in general use and they must be permeable to steam. The mouths of plastic bags should not be closed before autoclaving since this will reduce steam penetration.    

Chemical Disinfection

Chemical disinfection should be regarded as a first line defence especially in the case of discarded bench equipment, which should be followed by autoclaving or incineration. There is a rough spectrum of susceptibility of microorganisms to disinfectants. The most susceptible are vegetative bacteria, fungi, and enveloped viruses. Mycobacteria and non-enveloped viruses are less susceptible and spores are generally resistant. The most commonly used disinfectants in laboratory work are clear phenolics and hypochlorites. Aldehydes have a more limited application and alcohol mixtures are less popular. Iodophors and quaternary ammonium compounds (QAC) are more popular in the US than in the UK.

1. Clear Phenolics - these compounds are effective against vegetative bacteria (including mycobacteria), fungi, enveloped viruses (including Lassa and Marburg). They are inactive against spores and non-enveloped viruses. Most phenolics are active in the presence of considerable amounts of protein but are inactivated to some extent by rubber, wood, and plastics. They are not compatible with cationic detergents. Laboratory uses include discard jars and disinfection of surfaces. Clear phenolics should be used at the highest recommended concentration. Dilutions should be prepared daily and diluted phenolics should not be stored for more than 24 hours. Skin and eyes should be protected.

2. Hypochlorites - the active ingredient is chlorine, which is very effective against vegetative bacteria (including mycobacteria), spores, fungi, and both enveloped and non-enveloped viruses. Hypochlorites are considerably inactivated by protein and to some extent by natural non-protein material and plastics. They are not compatible with cationic detergents. Laboratory uses include discard jars and surface disinfection but they corrode some metals and thus care is necessary. They should not be used on the metal parts of centrifuges. For general purposes and discard jars, 2500 ppm is recommended. For spillages of blood and discard jars which may receive much protein, 10000 ppm should be used. The hypochlorites sold for laboratory applications in the UK contain 100000 ppm. Hypochlorites decay rapidly in use and diluted solutions should be replaced after 24 hours. Hypochlorites may cause irritation of skin, eyes, and lungs,

3. Aldehydes - formaldehyde (gas) and glutaraldehyde (liquid) are good disinfectants. They are active against vegetative bacteria (including mycobacteria), spores, fungi, and both enveloped and non-enveloped viruses. They are active in the presence of protein and are not very much inactivated by natural or man-materials or detergents. Formaldehyde is supplied either as a solid polymer, paraformaldehyde, or as a liquid, formalin. Both forms are heated to liberate the gas which is used for disinfecting enclosed spaces such as safety cabinets and rooms. Glutaraldehyde usually requires an activator, such as sodium bicarbonate. Most activators contain a dye so that the user can be sure that the disinfectant has been activated. Effectiveness and stability after activation varies with product and the manufacturer’s literature should be consulted. Glutaraldehydes are particularly useful in disinfecting metal surfaces as they do not cause corrosion. They may also be used in discard jars but they are comparatively expensive. Aldehydes are toxic and precautions must be taken when formaldehyde is used.

4. Alcohol and alcohol mixtures - alcohol and alcohol mixtures are effective, albeit slowly, against vegetative bacteria and enveloped viruses. They are not effective against spores, fungi and non-enveloped viruses. They are not inactivated by protein and other material or detergents. Alcohols and alcohol mixtures are useful for disinfecting surfaces. They are relatively harmless to skin but may cause eye irritation.

5. Quaternary ammonium compounds - they are cationic detergents that are effective against vegetative bacteria and enveloped viruses and some fungi. They are not active against mycobacteria, spores, and non-enveloped viruses. They are inactivated by protein and a variety of natural and plastic materials and soap. Their laboratory uses are therefore limited but they have the distinct advantages of being stable and non-corrosive to metals. They are usually used for cleaning surfaces and are also very popular in food hygiene laboratories because of their detergent nature. They are non-toxic and harmless to the skin and eyes.

6. Iodophors - like chorine compounds, these iodines are effective against vegetative bacteria (including mycobacteria), spores, fungi, and both enveloped and no-enveloped viruses. They are rapidly inactivated by protein, some natural and plastic substances and are not compatible with anionic detergents. Laboratory uses include discard jars and disinfecting surfaces. They can also be used for handwashing since they usually come with a detergent. Iodophors are relatively harmless to the skin by some eye irritation may occur.

Precautions in the use of disinfectants - As indicated above, some disinfectants have undesirable effects on the skin, eyes, and respiratory tract. Disposable gloves and safety spectacles, goggles, or a visor should be worn by anyone handling strong disinfectants. Full-face respirators should be worn when rooms are being fumigated with formaldehyde.

Discard Jars - discard jars should be robust and autoclavable. Glass jars should not be used because they are easily broken. Polypropylene beakers or jars are probably the most serviceable items. Wide-moth screw-capped polypropylene bottles or jars are better still. They are left open on the bench but are closed at the end of the working session. It is important that discard jars are filled frequently with known active dilutions of disinfectant, not overloaded with protein or floating articles. Laboratory supervisors should ensure that inappropriate articles are not placed in discard jars e.g. a 25ml pipette has no place in a small 1 litre jar. Funnels may be used to prevent splashing and aerosol dispersal. Discard jars should be washed with hot water before re-use. It is better still to autoclave the jar before washing. The contents of the discard jars should then be autoclaved and/or incinerated.

Re-usable pipettes - after use, re-usable pipettes should be completely immersed in disinfectant so that no air remains. They should remain in disinfectant for at least 18 hours before washing and/or autoclaving.    

 

Disinfection after accidents

There is an immediate need for disinfection after spillage, breakage or other accidents involving infectious material. The safety officer should draw up protocols for decontamination procedures after particular accidents. Action to be taken ranges from simple mopping up with disinfectant to decontamination of large items of equipment or of whole rooms. Appropriate equipment should be available for all such emergencies.

1. leakage of specimens - this is of particular concern when it happens in reception rooms with clerical staff. Saving the specimen, which may not be repeatable may be considered as important as protecting the staff. Leakages should be dealt with by a member of the laboratory staff. Disposable gloves should be worn and the offending container and any others which may be contaminated be placed on a tray which is then placed in a large plastic bag and removed to a laboratory, preferably a safety cabinet. The surface on which the leakage occurred should then be covered with paper towels over which the appropriate disinfectant is poured. This should be left for at least 30 minutes before the towels are removed. The area should then be swabbed with fresh disinfectant and left for a further 30 minutes before it is dried. The tray used should be flooded with disinfectant and left for several hours. Leakage into transport boxes should be treated in the same way except that the box can be autoclaved.

2. Simple spillages - simple spills should be covered promptly with a paper towel and disinfectant poured gently on the towel. The towel should removed after 30 minutes and the are swabbed with fresh disinfectant. If material is spilled in a safety cabinet, the fan should be left running while the spill is being dealt with. After a spillage, the safety cabinet should be disinfected by formaldehyde.

3. Serious spillages - the most serious spillages are those that involve cultures dropping on the floor and break. There is much splashing and a considerable amount of aerosol is dispersed. Laboratory workers should not bend down to inspect the damage. Instead, all people in the room should hold their breath and leave. Any possibly contaminated clothing should be removed. If that is not practical, then the affected area should be sponged with a disinfectant-detergent. Hypochlorites should not be used because it will bleach the fabric. The door should be closed and a warning sign posted onto it. Thirty minutes is a reasonable time to allow the aerosol to settle or clear. The safety officer should by this time have ascertained the possible nature of the organism involved and decide what respiratory or other protection is required when re-entering the room, which disinfectant to use, and the extent of the room decontamination procedures e.g. from mopping up to total decontamination of the room by fumigation

4. centrifuge accidents - breakage of tubes in a centrifuge can disperse large amounts of aerosols. Sealed buckets should be used for all Hazard Group 3 agents. The action to be taken is much the same as that for the breakage of a culture. After the room is considered safe to enter, the centrifuge buckets and rotor can be removed and autoclaved and the bowl disinfected.

5. Transport associated accidents - these may range from leakage of a pathological specimen in a hospital van to leakage or breakage of a culture in a public place, post office, public service vehicle or aircraft. In principle, all accidents can be treated as above for decontaminating surfaces and rooms. However, since the potential for the spread of infection is so great, it is essential to inform the senior medical microbiologist and environmental health officer at once. Protocols for dealing with these emergencies have been worked out by most health departments and have been published by the WHO.

 

Decontamination Procedures

1. Rooms - it may be necessary to disinfect a room after a spillage of infectious material, before a change of use or before building alterations or redecoration. Two methods may be used; wet disinfection and fumigation. Wet disinfection is only applicable if the floors do not allow liquids to leak into the rooms below and any remaining furniture or equipment is not likely to be damaged by disinfectant. The floor is mopped by a disinfectant and then left wet for at least 30 minutes. It may also be possible to disinfect the whole room with a sprayer. The operator should wear eye and face protection and if formalin is used, breathing apparatus should be worn. Fumigation of whole rooms is now not generally recommended because of health threats. If fumigation is carried out, then it is of utmost importance to ensure that there is no possible leakage from the room. Windows, ventilators, pipe runs and cracks should be sealed with masking tape. The operator must have full eye and face protection as well as a respirator. He should leave the room immediately after starting the reaction. The door should then be locked and sealed with masking tape and a warning notice be posted and the room left for 24 hours.

2. Equipment - it is desirable to have a regular procedure for decontaminating equipment that is to be serviced on site or returned to a manufacturer for any reason. Many companies require certificates that state specifically that the equipment is microbiologically safe. The interiors of incubators, refrigerators and similar cupboard-like equipment should be swabbed thoroughly with glutaraldehyde or alcohol-formalin mixture and left overnight. It should be washed out several times with clean water and dried. After decontamination, the equipment should be taped or otherwise secured so that it cannot be used and labelled. In the case of automated equipment, the effluent discharge tube should be taken at least 25 cm into he laboratory waste plumbing system or that it is trapped in a bottle. The instrument is then flushed through with a disinfectant recommended by the manufacturer.

3. Laboratory protective clothing - laboratory protective clothing should be autoclaved before it is sent for laundering or placed in "hot wash" bags which should not be opened in laundries until they have been exposed to very hot water.

   

Disposal of contaminated liquid waste

Small amounts of contaminated liquid waste may be poured into disinfectant but problems arise with large amounts such as urines, effluents from automated equipment, and sink and lavatory effluents from Maximum Containment laboratories. It is safe to pour most specimens down a sluice or deep sink which will join the public sewer. Sewage disposal plants deal quite effectively with most pathogens. The only hazard to the operator is from splashing and aerosol production when pouring and this should be done with care. Afterwards or at the end of the day, the sluice or sink should be flushed with disinfectant. If it is known or suspected that the urine contain Hazard Group3 pathogens, neat disinfectant (hypochlorite or phenolic) should be added to the urine. The effluents from automatic apparatus are unlikely to be very hazardous since microorganisms are likely to have encountered inimical chemicals on their way through the machine. The safe disposal of the various effluents from Containment Level 4 laboratories is a design consideration. The effluents are usually taken to one or one holding tanks where they are heated by steam for a predetermined time, then tested for the presence of pathogens before discharge into the sewers. Sometimes, chemical treatment is preferred.

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