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Tuberculosis Laboratory Biosafety Manual. Geneva: World Health Organization; 2012.

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Tuberculosis Laboratory Biosafety Manual.

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6Safety equipment

Safety equipment may be used to eliminate or reduce certain risks in TB laboratories (Table 4). Such equipment offers no assurance of protection unless the operator is competent and uses proper techniques. Equipment should also be tested regularly to ensure that it continues to perform safely.

Table 4. Safety equipment used to process specimens in TB laboratories, potential hazards and associated safety features.

Table 4

Safety equipment used to process specimens in TB laboratories, potential hazards and associated safety features.

6.1. Biological safety cabinets

Owing to their small size, droplet nuclei aerosols may be generated by certain laboratory procedures without the laboratory worker's knowledge; this may result in the inhalation of infectious agents or cross-contamination of work surfaces or materials. BSCs are designed to protect people and the environment from infectious agents and, depending on their classification, offer varying degrees of protection from contamination of specimens and cultures.

The HEPA filter in the exhaust system of a BSC effectively traps infectious organisms and ensures that only microbe-free exhaust air is discharged from the cabinet. A HEPA filter mounted in the BSC above the BSC work surface protects the surface and its materials from contamination. This is often referred to as product protection.

There are three classes of BSCs: I, II and III (corresponding to standards AS/NZS 2252.1:1994, AS/NZS 2252.2:1994, NSF/ANSI 49 – 2008 and EN 12469).18,19,20,21 According to the NSF/ANSI 49 – 2008 standard, class II BSCs have a variety of types (known as A1, A2, B1, B2); these are used to classify variations in airflow patterns, velocities, the position of the HEPA filter inside the cabinet, ventilation rates and exhaust methods.

6.1.1. Selecting a biological safety cabinet for a TB laboratory

The two types of BSCs described below are best suited for use in moderate-risk laboratories and in high-risk laboratories (TB-containment laboratories).

Class I

  • This type of BSC provides personal and environmental protection but does not offer product protection. This lack of product protection may contribute to increased contamination rates, especially when preparing and inoculating liquid cultures (see Figure 1).
Figure 1. Schematic diagram of a Class I biological safety cabinet A.

Figure 1

Schematic diagram of a Class I biological safety cabinet A. Front opening; B Sash; C Exhaust HEPA filter; D Exhaust plenum.

Class II

  • A Class II BSC offers personal, environmental and product protection, and, in type A2 models all biologically contaminated ducts are under negative pressure or are surrounded by negative-pressure ducts (see Figure 2). (This is the PREFERRED type of BSC.)
  • Class II type A1 BSCs are not a good choice because the ducts may become contaminated, and plenums have positive pressure relative to the room
  • Class II type B1 and type B2 BSCs must be hard-ducted to the outside; this means that the building's exhaust system must precisely match the airflow requirements specified by the manufacturer for both volume and static pressure. Certification, operation and maintenance of these types are therefore more difficult, so these BSCs are not recommended for any new TB laboratory facilities.
Figure 2. Schematic diagram of a Class II type A2 biological safety cabinet.

Figure 2

Schematic diagram of a Class II type A2 biological safety cabinet. A Front opening; B Sash; C Exhaust HEPA filter; D Supply HEPA filter; E Positive-pressure plenum; F Negative-pressure plenum.

BSCs should be equipped with HEPA filters that meet applicable international standards (for example, European norm standards EN12469 or United States NSF/ANSI Standard 49 – 2008).20,21

For all newly procured BSCs, Class II type A2 cabinets with a moveable sash are recommended.

A BSC should be selected primarily according to the type of protection needed: product protection or protection for personnel against the risk of infection. Selecting the correct type of BSC, installing it, using it properly, and annually certifying its operation are complex processes. It is highly recommended that these processes are performed by well trained and experienced professionals familiar with all aspects of BSCs.

BSCs should be connected to an uninterrupted power supply to ensure that staff have adequate time to complete a procedure in the event of a power outage.

Biosafety cabinets must undergo certification at the time of installation, whenever they are moved, and following any repairs or filter changes; they also require regular (annual) maintenance to ensure proper functioning. Delaying maintenance or using under-qualified personnel to conduct maintenance can put laboratory workers at risk. (See section 6.1.5.)

6.1.2. Class I biological safety cabinets

Class I BSCs work by drawing unfiltered room air in through a front opening, passing it over the work surface, and then expelling it through an exhaust duct.

Class I BSCs protect workers but do not protect work products (such as specimens or cultures) against contamination because unsterilized room air is drawn over the work surface.

Figure 1 is a schematic diagram of a Class I BSC. Room air is drawn in through the front opening at a minimum velocity of 0.38 m/s (NSF/ANSI).20 It then passes over the work surface and is expelled from the cabinet through the exhaust duct. The directional flow of air carries aerosol particles that may be generated on the work surface away from technicians and into the exhaust duct. The front opening allows the technician's arms to reach the work surface inside the cabinet while he or she observes the surface through a glass window. The window can also be fully raised to provide access to the work surface for cleaning or other purposes.

The air from the cabinet is expelled through a HEPA filter: (a) into the laboratory and then to the outside of the building through the building's exhaust system; or (b) to the outside through the building's exhaust system; or (c) directly to the outside.

6.1.3. Class II type A2 biological safety cabinets

Class II BSCs differ from Class I cabinets in that they allow only air from a HEPA-filtered (sterile) supply to flow over the work surface.

A Class II type A2 BSC is shown in Figure 2. An internal fan draws room air (supply air) into the cabinet through the front opening and then into the front intake grill. After passing through the grill, the supply air is drawn upwards and through a HEPA filter before flowing downwards over the work surface.

As the air flows downwards at about 6–18 cm above the work surface it splits so that approximately one half of the volume of the air passes through the front exhaust grill and the other half passes through the rear exhaust grill. Any aerosol particles generated at the work surface are immediately captured in this downward airflow and passed through the front or rear exhaust grills, thereby providing the highest level of product protection. The air is then discharged through the rear plenum into the space between the supply filter and exhaust filter located at the top of the cabinet. Owing to the relative size of these filters, 60–70% of the air recirculates through the supply HEPA filter back into the work zone; the remaining 30–40% passes through the exhaust filter into the room or outdoors.

Air from a Class II type A2 exhaust can be recirculated to the room or discharged to the outside of the building through a thimble connected to a dedicated duct; it must NOT be discharged through the building's exhaust system.

In a containment laboratory where air from the Class II BSC is recirculated to the room, a separate dedicated ventilation system is needed to ensure unidirectional flow of air into the laboratory with 6-12 ACH. Recirculating the exhaust air to the room has the advantage of lowering the energy costs of the building because heated or cooled air is not being passed to the outside environment.

6.1.4. Thimble connections

A thimble connection (see Figure 3) is used with Class II type A2 BSC that is ducted to the outside. The thimble fits over the cabinet's exhaust housing, sucking the air expelled from the cabinet into ducts that lead outside. A small opening (usually 5 cm wide) is maintained between the thimble and the cabinet's exhaust housing. This opening enables room air to be drawn into the exhaust ducting system. The capacity of the exhaust system must be sufficient to capture both room air and the cabinet's exhaust. The thimble must be removable or be designed to allow for operational testing of the cabinet. Generally, the performance of a thimble-connected BSC is not affected much by fluctuations in the building's airflow.

Figure 3. Schematic diagram of a thimble connection for a Class II type A2 biological safety cabinets ducted directly outside the laboratory.

Figure 3

Schematic diagram of a thimble connection for a Class II type A2 biological safety cabinets ducted directly outside the laboratory.

One advantage to using a thimble connection is that the BSC does not need any adjustments and the pressure in the room will remain nearly constant. To keep a controlled, constant, lowered pressure within the containment room, a damper control for the exhaust system is usually needed to enable the air flow through the thimble to be balanced with the exhaust capacity of the extractor fan placed at the end of the ducting.

Another advantage of using a thimble connection is that in case of power outages, the air flowing back into the room where there is lowered pressure will pass nearly exclusively through the thimble air intake, and not wash off bacteria from the HEPA filter. Installing a valve to prevent backflow in the duct ensures that air flowing in will travel through the clean-air intake.

6.1.5. Using biological safety cabinets in the laboratory

Location

The integrity of the directional air inflow is fragile and can be easily disrupted by air currents generated by people walking close to the BSC, by open windows or air-supply registers, and by the opening and shutting of doors. Ideally, BSCs should be situated as recommended by the manufacturer in a location away from traffic and from potentially disruptive air currents. Whenever possible, a clearance of 30 cm should be provided behind and on each side of the cabinet to allow easy access for maintenance. A clearance of 30–35 cm above the cabinet may be required to accurately measure air velocity across the exhaust filter, and to change exhaust filters.

Operators

If BSCs are not used properly, their protective benefits may be greatly diminished; in some instances improper use can even result in increased risk to the laboratory worker. Written protocols, as well as a biosafety manual, should be issued to laboratory staff and they should sign a form to confirm that they have read and understood the required protocols. All individuals working in BSCs should be observed to ensure they follow correct working practices before they routinely perform testing in the BSC. Operators need to maintain the integrity of air flowing through the front opening when moving their arms into and out of cabinets. They should move their arms slowly and ensure they are perpendicular to the front opening. Staff should wait about 2 minutes after placing their hands and arms inside the BSC before they begin manipulating materials; this will allow the airflow within the cabinet to adjust and the air to sweep the surface of their hands and arms. The number of movements made across the front opening should be minimized by placing all necessary items into the cabinet before beginning manipulations.

Material placement

The front intake grill of Class II BSCs must not be blocked with paper, equipment or other items. It is recommended that all work be performed on disinfectant-soaked absorbent towels arranged to capture splatters and splashes. All materials should be placed as far back in the cabinet as practical – that is, towards the rear of the work surface – without blocking the rear grill. Aerosol-generating equipment (such as vortexes and centrifuges should be placed towards the rear of the cabinet. Bulky items (such as biohazard bags and discard containers) should be placed to one side of the interior of the cabinet. Active work should flow from clean areas to contaminated areas across the work surface. Paperwork should never be placed inside BSCs. The cabinet must not be overloaded because overloading may affect the efficiency of the airflow (see Figure 4).

Figure 4. Typical layout for working from clean areas to dirty areas within a Class II biological safety cabinet.

Figure 4

Typical layout for working from clean areas to dirty areas within a Class II biological safety cabinet. Clean materials are placed to the left of the cabinet; samples are inoculated in the centre of the cabinet; and contaminated pipettes and other materials (more...)

Ultraviolet lights

Ultraviolet lights are not recommended in BSCs used in TB laboratories.

Open flames

Open flames must be avoided in BSCs because heat disrupts the patterns of airflow within the cabinets. To sterilize bacteriological loops, microincinerators or electric furnaces are available, and their use is preferable to open flames. The use of disposable loops and disposable transfer pipettes is preferred.

Spills

A copy of the laboratory's protocol for handling spills should be posted, read and understood by all laboratory staff. When a spill occurs inside a BSC, clean up should begin immediately and the cabinet should continue to operate. An effective disinfectant should be used and applied in a manner that minimizes the generation of aerosols. All materials that come into contact with the spilled agent should be disinfected and disposed of properly.

Certification

The functional operation and integrity of each BSC should be certified to national or international performance standards at the time it is installed, following any relocation with the laboratory, and regularly thereafter (at least annually) by qualified service technicians, according to the manufacturer's specifications. An evaluation of the effectiveness of the cabinet's containment capability should include tests of the cabinet's integrity; tests for HEPA filter leaks; assessments of the down flow velocity profile, face velocity, negative pressure and ventilation rate, airflow smoke pattern, and alarms and interlocks.

The velocity of air flowing through the front opening into a BSC should meet the manufacturer's specifications. Optional tests may also be conducted for electrical leakage, lighting intensity, ultraviolet light intensity, and noise level and vibration. Special training, skills and equipment are required to perform these tests, and it is highly recommended that they are undertaken by an experienced professional. The professional should be familiar with and trained in all aspects of BSCs.

Cleaning and disinfecting the work area

When work is completed, all items within a BSC, including equipment, should have surfaces decontaminated and be removed from the cabinet.

The interior surfaces of BSCs should be decontaminated before and after each use. Work surfaces and interior walls should be wiped with a disinfectant that will kill any microorganisms that might be found inside the cabinet. At the end of the workday, the final surface decontamination should include wiping down the work surface, and the sides, back and interior of the glass. A second wiping with sterile water is needed when a corrosive disinfectant, such as bleach, is used.

Before it is switched off, the BSC should be left to run for 15 minutes after work is completed in order to purge the atmosphere inside.

Decontamination

BSCs must be thoroughly decontaminated before filters are changed and before the cabinet is moved; decontamination must include plenums and filters. See standard NSF/ANSI 49 – 2008 for procedures and details of decontamination.20 Decontamination should be performed by a qualified professional.

Alarms

BSCs can be equipped with one of two audible alarms. Sash alarms are found only on cabinets with sliding sashes. The alarm sounds when the laboratory worker has moved the sash to an improper position. When this alarm sounds, the sash must be returned to the proper position. Airflow alarms indicate a disruption in the cabinet's normal airflow pattern. This alarm represents an immediate danger to the worker or product. When an airflow alarm sounds, work should cease immediately and the laboratory manager should be notified. Manufacturers' instruction manuals should provide further details about how to address this type of alarm. Training in the use of BSCs should include information on how to respond to this type of alarm.

6.2. Centrifuges with safety buckets

During the centrifugation process, aerosols may be produced. Consequently, safety measures must be strictly followed when operating the centrifuge.

During centrifuge operation, the lid must be fully sealed. The use of a wide nonporous seal will ensure that the lid is closed tightly. The lid must not be opened until the rotor has stopped completely. Appropriate rotors have safety caps for each slot. The caps for individual buckets and tubes must be closed properly before the centrifuge is operated. To contain aerosols, individual sealed centrifuge buckets should be loaded and unloaded in a BSC. For processing TB cultures, refrigerated centrifuges with swinging buckets are recommended.

When using a micro-centrifuge for DNA extraction, a safety rotor is needed with a sealed lid; the micro-centrifuge should be loaded and unloaded inside a BSC.

Centrifuges must be inspected periodically for wear and tear; and maintenance must follow the manufacturer's specifications.

6.3. Autoclaves

In general TB laboratories that perform diagnostic tests, an autoclave that uses saturated steam under pressure is the most efficient means of sterilizing instruments, glassware and media solutions; it is also used for decontaminating biological material (such as mycobacterial cultures). Two factors are essential for an autoclave to function optimally: (1) all of the air in the chamber should be replaced by steam; and (2) the temperature must be 121 °C.

Autoclaves should be located away from the main laboratory working area because they may be noisy, hot and release steam. An autoclave used to decontaminate infectious material, should have an exhaust air valve equipped with a bacterial filter. The autoclavable sterile filter should consist of a filter cartridge with a membrane (pore size 0.2 μm) incorporated into a pressure-resistant housing; the filter should be easy to replace. The filter is automatically sterilized during each sterilization process. An autoclave MUST be available in each facility where TB cultures are performed and should ideally be placed within the TB-containment laboratory.

The manufacturer's instructions for operating and cleaning the autoclave must be followed at all times.

Copyright © World Health Organization 2012.

All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: tni.ohw@sredrokoob). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site (http://www.who.int/about/licensing/copyright_form/en/index.html).

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