Part I: Organisation and management
laboratory hygiene and safety

With the escalation of tuberculosis world-wide, driven by the HIV epidemic and aggravated by the emergence of multidrug-resistance, renewed concern has arisen about safety in tuberculosis laboratories.

Studies have shown that the risk of tuberculosis infection is three to five times higher for laboratory workers when compared to administrative staff or the general community, depending on the type of laboratory work done. The potentially dire consequences of becoming infected with multidrug-resistant strains of M. tuberculosisand the increasing proportion of persons infected with HIV (which include laboratory workers) add a new dimension to the problem and emphasise the importance of strict adherence to safety precautions in the laboratory.

M. tuberculosis is included in Risk Group III in the 1983 WHO classification of risk, along with other micro-organisms most likely to infect laboratory workers by the airborne route. The number of tubercle bacilli required to initiate infection is low, the infective dose being less than 10 bacilli. Infective particles in the laboratory are usually derived from moist droplets discharged into the air by procedures liberating aerosols. When aerosolised material dry out droplet nuclei of 1Fm to 5Fm in size are formed creating infective particles which may remain in the air for long periods of time.

Safety precautions in tuberculosis laboratories must involve measures to:

• minimise the production and dispersal of aerosols and infective particles
• prevent laboratory workers from inhaling airborne particles and
• prevent infection by accidental inoculation and ingestion

The focus of bio-safety in the tuberculosis laboratory should be on primary containment measures which are aimed at protecting laboratory staff and the immediate environment. Appropriate ventilation should flow from clean to contaminated areas. In peripheral laboratories in tropical countries windows may be necessary; however, these should be located in such a way that air currents do not pass over the area of smear preparation in the direction of the laboratory worker preparing the smears. In culture laboratories air should be continuously extracted to the outside of the laboratory at a rate of six to twelve air changes per hour. Supply and exhaust air devices should be located on opposite walls, with supply air provided from clean areas and exhaust air taken from less clean areas. Exhaust air must be discharged directly to the outside of the building to be dispersed away from air intakes.

Patients with tuberculosis are increasingly co-infected with HIV and their specimens may either be contaminated with blood or be a primary source of the virus, eg. blood or bone marrow specimens. Attention should also be focused on the increased risk of laboratory workers contracting tuberculosis should they be or become infected with HIV.

Safety in the tuberculosis laboratory must start at the administrative level. It is an administrative responsibility to ensure that laboratory staff are:

• trained properly in safe laboratory procedures
• informed of especially dangerous techniques and procedures that require special care
• provided with adequate safety equipment and clothing
• prepared for prompt corrective action following a laboratory accident
• educated about their increased risk of acquiring tuberculosis should they be or become HIV positive
• monitored regularly by medical personnel

The laboratory worker is responsible for:

• following established laboratory policy
• accepting the responsibility for correct work performance to assure the safety of fellow  workers
• using appropriate safety equipment
• accepting the responsibility for maintaining a health lifestyle

Procedural hazards
Inhalation hazards
Most infections in the tuberculosis laboratory can be attributed to the unrecognised production of potentially infectious aerosols containing tubercle bacilli. Many microbiological techniques generate aerosols, eg. when bubbles burst or when liquids are squinted through small openings or impinge on surfaces. Large (>5Fm) aerosolised droplets settle rapidly to contaminate skin, clothing and counter tops; however, the most dangerous aerosols are those that produce droplet nuclei - tiny dry particles less than 5Fm in size that may contain one or more viable tubercle bacilli. Droplet nuclei can float in the air almost indefinitely and are inhaled and trapped in the lung alveoli where they initiate infection.

The following microbiological activities generate aerosols:

• Collecting sputum  specimens from coughing patients
  Tuberculosis suspects are sometimes referred directly to the laboratory for sputum collection. This practice exposes laboratory workers to a high risk of infection by aerosols produced during collection procedures. Precautions to lower this risk include instructing tuberculosis suspects to cover their mouths while coughing, standing behind (and not in front of) coughing individuals and collecting specimens  outdoors where aerosols are diluted and sterilised by direct sunlight.
   
• Adding decontamination solutions
  Decontamination solutions should be added gently and never be mixed while  containers or tubes are open. After the digestion / decontamination procedure, pour the  supernatant fluid through a funnel into a splash-proof container to minimise both aerosol  production and contamination of the work surface. Gently discard the fluid down the side  of the funnel into an appropriate disinfectant solution (eg. 5% phenol).
   
• Working with bacteriological loops
  Loops should be handled gently and vigorous spreading of inocula on medium  or from cultures should be avoided. Loops containing infectious material should first be  cleaned by rotating them in a 250ml screwcap flask containing 70% alcohol and washed sand. They can then be sterilised in a Bunsen flame, the alcohol causing rapid incineration of  residual debris.
   
• Pipetting
  Never pipette by mouth. Not only is there a danger of aspirating infectious material, but aerosols are created when fluid is alternately sucked and expelled through the pipette. With the nose directly over the open container of infectious  material, the aerosols have direct access to the lungs.
  
  Pipettes should be drained gently and not blown out violently, otherwise  the last drop will form bubbles which burst and create aerosols. Pasteur pipettes are particularly likely to generate bubbles.
   
• Centrifugation
  The recommended type of centrifuge for tuberculosis laboratories is a floor model with a lid and fixed angle rotor which contains sealed centrifuge buckets or aerosol-free carriers. Centrifuges should preferably be fitted with an electrically operated safety catch which prevents the lid from being opened while the rotor is  spinning.
  
  Tubes should always be capped during centrifugation and balanced within  centrifuge buckets to avoid breakage. If centrifuge tubes leak, crack or shatter during centrifugation, an invisible cloud of potentially infectious droplet nuclei may be  released.
   
• Pouring into disinfectant
  When infectious material is poured into disinfectant it may splash and  create aerosols, while contaminating surrounding surfaces. Use a funnel with its end  beneath the surface of the disinfectant and gently discard the material down the side of the funnel.

Ingestion hazards
Tuberculosis materials may be ingested by direct aspiration through mouth pipetting and by putting into the mouth fingers and objects that have been contaminated while in contact with the laboratory bench. Fingers may also become contaminated by the outside of specimen containers. Routine disinfection of containers before processing are recommended and frequent hand washing should be routine practice.

Inoculationhazards
Needle stick accidents are not uncommon and hypodermic needles should never be used as a substitute for pipettes. Cuts from contaminated glassware may also occur and touching of broken glassware must be avoided. Glass Pasteur pipettes are the most dangerous and glassware should be substituted with plastic materials whenever possible.

Laboratory hygiene

• Entry to the laboratory should be restricted to laboratory staff only
• Eating, drinking, smoking or applying make-up must be  prohibited in the laboratory
• Mouth-pipetting, licking labels and sucking pencils should not be allowed
• Hands must be washed with a suitable bactericidal soap upon  entering the laboratory, after handling potentially contaminated specimen containers, after any bacteriological procedure, after removing protective clothing and before leaving  the laboratory. Disposable paper towels should be used for hand drying
• No cleaning, service or checking of equipment should be  allowed unless a trained technical or professional person is present to ensure adequate  safety precautions
• All surfaces and equipment within the laboratory should be regarded as potentially infectious and should be cleaned regularly by appropriate means.  Floors should not be waxed or swept but should be mopped regularly to limit dust formation

Disinfectants
The temporal killing action of disinfectants depends on the population of organisms to be killed, the concentration used, the duration of contact and the presence of organic debris.

The proprietary disinfectants suitable for use in tuberculosis laboratories are those containing phenols, hypochlorites, alcohols, formaldehydes, iodophors or glutaraldehyde. These are usually selected according to the material to be disinfected. Sweet-smelling ?anti-septics? should not be used. It is incorrect to assume that a disinfectant which has general usefulness against other micro-organisms is effective against tubercle bacilli. A number of commercially available disinfectants have no or little mycobactericidal activity, while quaternary ammonium compounds are not effective at the recommended concentrations.

Disinfectant solutions should be prepared fresh each day and should not be stored in diluted form because their activity will diminish.

Phenol should be used at a concentration of 2% to 5% and contact time should be 15-30 minutes, depending on the type and volume of material to be disinfected. Phenol is useful in soaked paper towels to cover working surfaces. This minimises spatter and aerosol formation in the event of spilling.

Hypochlorite should be used at concentrations between 1% and 5%, with a contact time of 15-30 minutes, depending on the type and volume of material to be disinfected. Hypochlorite solutions (5%) are useful for the disinfection of material containing organic debris because of their digesting action.

Glutharaldehyde does not require dilution but an activator (provided separately by the manufacturer) must be added. Glutaraldehyde is usually supplied as a 2% solution, while the activator is a bicarbonate compound. Glutaraldehyde is useful for decontaminating bench surfaces and glassware. The activated solution should be used within two weeks and discarded if turbid.

Alcohols, usually 70% ethanol (methylated spirits) or propanol is used in alcohol-sands baths and for decontaminating benches and surfaces. It should also be used instead of water to balance centrifuge tubes. When hands become contaminated, a rinse with 70% isopropyl alcohol followed by thorough washing with soap and water is effective.

Iodophor preparations should be used at concentrations of 3% to 5% and contact time should be 15-30 minutes, depending on the type and volume of material to be disinfected. Iodophors are useful for mopping up spills and for handwashing.

All of the above disinfectants are toxic and undue exposure may result in respiratory distress, skin rashes or conjunctivitis. However, used normally and according to the manufacturers? instructions, they are safe and effective.

Essential safety equipment and supplies
Biological safety cabinets
The single most important piece of laboratory safety equipment needed in the tuberculosis culture laboratory is a well-maintained, properly functioning biological safety cabinet (BSC).

BSCs use high efficiency particulate air (HEPA) filters in their exhaust and/or air supply systems. HEPA filters remove particles $3Fm (which essentially includes all bacteria, spores and viruses) with an efficiency of 99.97%.

Two types of cabinets can be used in tuberculosis culture laboratories: One is a Class I negative-pressure BSC that draws a minimum of 75 linear feet of air per minute (22.86 meter per second) across the front opening and exhausts 100% of air to the outside. Class I BSCs provides protection to the user (ie. laboratory worker) but not to the product (ie. cultures for tubercle bacilli). Strict precautions are, therefore necessary to avoid contamination of culture media. The other BSC is a Class II vertical laminar flow cabinet that blows HEPA filtered air over the work area. Because cabinet air has passed through the HEPA filter, it is contaminant free and may be circulated back into the laboratory (Type A) or ducted out of the building (Type B).

The airflow through the BSC is adjusted by the manufacturer to provide at least 75 linear feet per minute (22.86 meter per second) and should be tested and re-certified once a year by trained personnel. Regular checks (eg. quarterly, or more often under dusty conditions) on the airflow should be made with an anemometer. If the airflow is appreciably diminished this indicate that the filters have become clogged and that the BSC needs to be decontaminated.

The following safety precautions need to be observed when working within a BSC:

• Use proper microbiological techniques to avoid splatter and aerosols. This will minimise the potential for staff exposure to infectious materials manipulated within the cabinet. As a general rule, keeping clean materials at least 12cm away from aerosol-generating activities will minimise the potential for cross-contamination
• Do not hold opened tubes or bottles in a vertical position and recap or cover them as soon as possible. This will reduce the chance for cross-contamination
• Do not use open flames in the BSC. This creates turbulence which disrupts the pattern of  air supplied to the work surface. Small electric furnaces are available for  decontaminating loops and are preferable to an open flame inside the BSC
• Use an appropriate liquid disinfectant in a discard pan to decontaminate materials  before removal from the BSC. Introduce items into the pan with the minimal splatter and allow sufficient contact time before removal. Alternatively, contaminated items may be placed into an autoclavable disposal bag within the BSC. Water should be added to the bag prior to autoclaving to ensure steam generation during the autoclave cycle

BSCs have to be decontaminated at least once a year and the filters replaced by creating a formaldehyde vapour which will kill all tubercle bacilli trapped in the filters. Always decontaminate the BSC before HEPA filters are changed or internal repair work is done. The most common decontamination method uses formaldehyde gas created by adding potassium permanganate to formaldehyde liquid, as indicated by Diagram 1.

Protective clothing
Suitable clothing must be worn while working in the laboratory:

• Coats and gowns should wrap across the body and cover the upper chest and  neck. Sleeves should be wrist length
• Gloves should be worn when handling potentially infectious materials or when  touching potentially infectious surfaces or equipment. Gloves also guard against infection through cuts or abrasions on the hands. Disposable gloves should not be re-used
• Industrial face masks designed to filter >95% of particles ranging from 1-5Fm could be worn during aerosol-producing procedures,  especially in culture laboratories. Masks should be discarded after eight hours of use

Protective clothing should be removed before leaving the laboratory and should be placed in covered containers or laundry bags before being washed or discarded.

Coping with a laboratory accident
Laboratory safety does not just happen. It is the result of:

• recognising that accidents can and will occur
• formulating a plan of action to neutralise the potential harmful effects of an accident as rapidly and effectively as possible
• discussing ways to minimise and prevent accidents from occurring

The best defence against a laboratory accident is a well-thought-out plan to neutralise its effects as quickly and effectively as possible. No accident should be considered insignificant; however, assessment of the seriousness of each accident is necessary to determine the most appropriate course of action.

• Be prepared for an accident by having the following readily accessible in or near areas where accidents are  most likely to occur:
  • a supply of paper towels or large cloths
  • a wide-mouthed (to facilitate rapid pouring) container of  disinfectant
  • a supply of industrial face masks capable of filtering particle sizes between 1Fm and 5Fm

A fogging machine is useful to quickly disperse disinfectant into a room in the case of an accident. The mist released by the machine rapidly saturates the air, causing the dangerous droplet nuclei to settle. An appropriate disinfectant used in the fogging machine will decontaminate potentially infectious droplet nuclei as they settle on floors and bench tops. The fogging machine should always be kept ready with an adequate volume of freshly-prepared disinfectant.

Accidents that occur in tuberculosis laboratories may be divided into two types: those that generate limited aerosols and those that produce a large volume of potentially infected aerosols. A limited aerosol may, for example, be created by breaking a single culture tube of egg medium or spilling the contents of a sputum specimen. In these instances, the solid medium and thick mucoid nature of the sputum specimen greatly limit large numbers of tubercle bacilli from being aerosolised. A plan of action for limited aerosol accident is presented in Diagram 2.

A large volume of potentially infectious aerosols may be generated by breaking one or more tubes of liquid containing a high concentration of tubercle bacilli, eg. bacterial suspensions or unbalanced centrifuge tubes. A plan of action for a large volume aerosol accident is presented in Diagram 3.

Accidents within the BSC
In the event of any spillage within the BSC, the cabinet should not be switched off. A plan of action for a limited aerosol accident in the BSC is presented in Diagram 4. A plan of action for a large volume aerosol accident in the BSC is presented in Diagram 5.