Arthropod Containment Level
	1	2	3
		Standard
Location of arthropods	Furniture and incubators containing arthropods are located in such a way that accidental contact and release is minimized.  This may be achieved by locating arthropods out of the flow of general traffic, avoiding hallways, or placing them in closets.	Furniture and incubators containing arthropods are located in such a way that accidental contact and release by laboratorians, custodians, and service persons is unlikely.  This may be achieved by locating arthropods in dedicated rooms, closets, incubators located out of the traffic flow etc.	Furniture and incubators containing arthropods are located in such a way that accidental contact and release by laboratorians, custodians, and service persons does not occur.  This is usually achieved by locating arthropods in dedicated rooms, wings or suites in  incubators located out of the traffic flow in areas of the building dedicated to BSL-3 activities.
Supply Storage	The area is maintained to allow detection of escaped arthropods.  For example, material unrelated to arthropod rearing and experimentation (e.g., plants, unused containers, clutter) that provide breeding sites and refugia are minimized.	The area is maintained to allow detection of escaped arthropods.  Equipment and supplies not required for operation of the insectary is minimized to allow detection of escaped arthropods.  If supplies must be kept within the insectary, they are located in a designated area and not on open shelves. It is recommended that a closed storage room, cabinets with tight-fitting doors or drawers be used. Doors and drawers are opened only for access.	Equipment and supplies not absolutely required for ongoing ACL-3 work are removed from the insectary after appropriate decontaimination.  Those present are located in a designated area and not on open shelves. It is recommended that a closed storage room, cabinets with tight-fitting doors or drawers be used.  Doors and drawers are only opened for access.
General arthropod elimination	Accidental sources of arthropods from within the insectary are eliminated.  This may be accomplished by cleaning work surfaces after a spill of materials including soil or water that might contain viable eggs.  Pools of water are mopped up immediately.	ACL-1	In addition to measures for general arthropod elimination within the insectary, materials wiped or mopped up are autoclaved before disposal.  Only persons trained and equipped to work with arthropods and BSL-3 agents clean up spills.
Primary container cleaning and disinfestation	Arthropods do not escape by inadvertant disposal in primary containers.  Cages and other culture containers are appropriately cleaned to prevent arthropod survival and escape (e.g., heated to over the lethal temperature or killed by freezing).	In addition to cleaning cages and culture containers to prevent arthropod escape as in ACL-1, containers are autoclaved if used for infected material.  Autoclaving or incineration of primary containers is recommended for containers holding uninfected material.	Care is taken to disinfest primary containers in a manner that does not create aerosols.  All primary containers are autoclaved or incinerated.
Primary Container Construction	Cages used to hold arthropods effectively prevent escape of all stages.  Screened mesh, if used, is durable and of a size appropriate to prevent escape.  Non-breakable cages are recommended.  Bags, rearing trays etc. effectively prevent leakage and escape.	Cages used to hold arthropods are non-breakable and screened with mesh of a size to prevent escape.  Containers are preferably autoclavable or disposable.   Openings designed to prevent escape during removal and introduction of arthropods are recommended.	Cages used to hold arthropods are non-breakable and screened with mesh of a size to prevent escape. Containers are autoclavable or disposable.  Openings are designed to prevent escape during removal and introduction of arthropods.  Disposable containers are recommended.
Disposal of Arthropods	Living arthropods are not disposed of inadvertently.  All wastes from the insectary (including arthropod carcasses, and rearing medium) are transported from the insectary in leak-proof, sealed containers for appropriate disposal in compliance with applicable institutional or local requirements.  All stages of arthropods are killed before disposal.  Autoclaving or incineration of material infected with a non-pathogen is recommended.   Material may be killed with hot water or freezing before flusing down drains.	In addition to standard ACL-1 disposal practices, autoclaving or incineration of arthropod materials is recommended.  Infected arthropods are autoclaved or incinerated.	In addition to ACL-2 disposal practices, the outer surfaces of containers are decontaminated prior to moving the material. All arthropod waste materials are autoclaved or incinerated.
Isolation of Un-infected Arthropods		Spread of agents to uninfected arthropods is prevented.  Generally this is accomplished by isolating infected material in a separate room.	Where possible, only arthropods requiring ACL-3 procedures are housed in the ACL-3 insectary.  If it is necessary to house ACL-2 or lower arthropods in the ACL-3 insectary, all procedures and practices must meet the ACL-3 standards.
Primary Container Identification and labeling	Arthropods are identified adequately.  Labels giving species, strain/origin, date of collection, responsible investigator, etc. is firmly attached to the container (and cover if removable). Vessels containing stages with limited mobility (e.g., eggs, pupae, hibernating adults) are securely stored.	ACL-1	ACL-1
Prvention of Accidental Dispersal on Persons or via Sewer	Personnel take appropriate precautions to prevent transport or dissemination of arthropods from the insectary on their persons or via the sewer.	Before leaving the insectary and after handling cultures and infected arthropods, personnel wash their hands, taking care not to disperse viable life stages into the drainage system.   No infected material is disposed of through the sewer.  If uninfected materials are disposed of via the sewer,  all material is destroyed by heat or freezing and preferably by autoclaving or incineration.  Air curtains are recommended as appropriate.	Before leaving the insectary and after handling cultures and arthropods, personnel wash their hands, taking care not to disperse viable life stages into the drainage system.   No material is disposed of through the sewer.  Uninfected material may be destroyed by heat or freezing if followed by autoclaving or incineration.
Pest exclusion program	A program to prevent the entrance of wild arthropods (e.g., houseflies, cockroaches, spiders) and rodents effectively precludes predation,  contamination, and possible inadvertent infection.	ACL-1	ACL-1
Escaped arthropod monitoring	Investigators assess whether escapes are occurring.  An effective arthropod trapping program is recommended to monitor the escape prevention program.	Investigators assess whether escapes are occurring by instituting an effective arthropod trapping program to monitor the escape prevention program. Oviposition traps, ground-level flea traps, oil-filled channels surrounding tick colonies, light traps for mosquitoes etc. are considered.   Particularly in the case when exotic arthropods are used, exterior monitoring is considered.  Records of exterior captures are maintained.	Additional measures are taken to measure the effectiveness of the arthropod trapping program and these are documented.  As part of the commissioning process of a new facility the physical integrity and security practices could be tested by a simple release-recapture study.  A known number of uninfected arthropods would be released and then attempts made to account for all of them. Such an experiment is described by Hunt and Tabachnick (6).  Exterior and within-building monitoring is considered.  Records of exterior captures are maintained.
Source and Refugia Reduction	Refugia and breeding areas are reduced as appropriate.  Furniture and racks are minimized and can be easily moved to permit cleaning and location of escaped arthropods.	Refugia and breeding areas are eliminated.  Furniture and racks are minimized and can be easily moved to permit cleaning and location of escaped arthropods.  Equipment in which water is stored or might accumulate (e.g., humidifiers) are screened to prevent arthropod access, or contain chemicals to prevent arthropod survival.	ACL-2
Microbiological  and Medical Sharps	Syringes that re-sheath the needle, needle-less systems, and other safe devices are used when appropriate.  Plastic-ware is substituted for glassware whenever possible.	ACL-1	Sharps are stringently limited and use is justified only when alternatives are not available.
Arthropod Sharps	Equipment such as needles, probes, dissecting tools and syringes are minimized.	In addition to minimizing arthropod sharps,  these are restricted for use in the insectary if infected materials are used.	In addition to minimizing arthropod handling shaprs, these are restricted for use in the insectary regardless of infection status of material handled.
Routine Decontamination		Equipment and work surfaces in the insectary are routinely decontaminated with an effective chemical or by radiation (e.g., heat) after actual or potential contact with an infectious agent, and especially after overt spills and splashes of viable materials (including soil or water that might contain infectious agents or eggs).	ACL-2
Notification and Signage	Persons entering the area are aware of the presence of arthropod vectors.	Persons entering the area are aware of the presence of arthropod vectors.  If infected material is present, a BSL-2 biohazard sign is posted on the entrance to the insectary listing all species handled within and is updated whenever new species are introduced or pathogenic infectious agents are present.  The hazard warning sign identifies the arthropod species, agent(s) known or suspected to be present, lists the name and telephone number of the responsible person(s), and indicates any special requirements for entering the insectary (e.g., the need for immunizations or respirators).	ACL-2 measures are implemented with BSL-3 signage.
Procedure design		All procedures are carefully designed and performed to minimize the risk of arthropod escape	All procedures are carefully performed to prevent arthropod escape and the creation of aerosols or splatters.  Protocols are practiced with un-infected arthropods / animals and modified before implementation.
Safety manual		A safety manual is prepared and adopted.  The manual contains emergency procedures, standard operating procedures, waste disposal and other information necessary to inform personnel of the methods for safe maintenance and operation of the insectary.	ACL-2
Training		Laboratory personnel are advised of special hazards and are required to follow instructions on practices and procedures contained in the safety manual.  Adherence to established safety procedures and policies is made a condition of employment and is part of the annual performance review of every employee.  Personnel receive annual updates and additional training as necessary for procedural or policy changes.  Records of all training are maintained.	The training required for laboratory personnel under ACL-3 is more detailed and extensive and BSL-3 certification is required if infected materials are handled.
Medical Surveillance		An appropriate medical surveillance program is in place.  All personnel receive appropriate immunizations or tests for the agents handled or potentially present.  When appropriate, a serum surveillance system is implemented (see BMBL for guidance).    Personnel are aware of the symptoms of infection and are aware of procedure to follow in reporting these.  In general, persons who may be at increased risk of acquiring infection, or for whom infection may be unusually hazardous (e.g., immunocompromized) are not allowed in the insectary unless special personal protection procedures are in place to eliminate extra risk.	In addition to the measures required for medical surveillance under ACL-2, assessment is made by the occupational health physician for persons who may be at unusual risk.
Access Restrictions		Routine access is limited to trained persons and accompanied guests.  Service persons are made aware of the hazards present and the consequences of arthropod release and contact with agents that may be present.	The insectary director limits access to the insectary to the fewest number of individuals possible.  Personnel who must enter the insectary for program or service purposes when work is in progress are accompanied by trained laboratorians and are advised of the potential hazard to themselves, co-workers and the potential consequences of arthropod release.
Speical Arthropod Handling Containers and Areas		Infected arthropods are prevented from release into the laboratory area.  This may be accomplished by secure glove boxes, biosafety cabinets, custom handling trays etc.  These may vary from BSL recommendations insofar as necessary to safely contain both the arthropod and any agent.  Such modifications should be made only in consultation with biosafety experts.  A dedicated area for handling infected material is recommended.  This is preferably a separate cubicle, walk-in incubator, or screen room.	All work is done in a primary barrier.  Appropriate biological safety cabinets, other physical containment devices, and/or personal protective equipment are used whenever conducting procedures to infect arthropods with BSL-3 agents, or when handling arthropods. Appropriate designs will consider the life history and behavior of the arthropod and may differ from that required by the agent alone.  Such modifications should be made in consultation with biosafety experts.  Manipulation of arthropods and, for example, rearing of transovarially infected immature stages are performed in a designated area. SALS (5) suggests “a separate room or double screened area that is separated from the main insectary by rooms having two screened or solid doors that open inward and closing automatically”
Safe Transport in the Laboratory		All infectious and potentially infectious samples are collected, labeled, transported, and processed in a manner that contains and prevents transmission of the agent(s). Transfer of arthropods between manipulation and holding areas is in non-breakable secure containers.	ACL-2
		Special
IACUC and IBC Approval	IACUC approval is required for use of vertebrate animals used as hosts.	IBC approval is required and IACUC if vertebrates are used as hosts.	ACL-2
Housing of Non-Arthropod Animals	Other animals not necessary for culture of the arthropods are not accessible to the arthropods.  Animals used as a hosts or blood sources may be housed within the insectary but are adequately protected from access by escaped arthropods.  Protocols for vertebrate animal use are approved by the IBCommittee and IACUC.	Other animals are not accessible to the arthropods.  Animals used as hosts or blood sources generally are not housed with arthropods.  If present they are adequately protected from access by escaped arthropods and protocols are approved by the IBC and IAUCUC.	ACL-2
Containment During Blood-Feeding	Arthropods fed on host animals are prevented from accidental transfer to host cages.  When handling/removing animals after exposure to arthropods, precautions must be taken to prevent arthropod escape through screens, covers, and by flying.  Host animals are inspected closely (e.g., concealment in fur, ears, crevices), and the primary container is sufficiently robust to prevent escape during feeding.	Recommendations for ACL-1 containment of arthropods during blood-feeding are more stringently assured by special practices and container design.	Recommendations for ACL-1 containment of arthropods during blood-feeding are strictly assured by special practices and container designs that prevent escape of arthropods.
Blood Source	The blood source is considered as a source of inadvertent arthropod infection and transmission.  Measures are implemented to prevent such an event.  Use of sterile blood or blood from sources known to be pathogen-free is recommended.  In contrast, use of blood from animals or humans whose disease status is uncertain is to be avoided.	ACL-1	ACL-1
Escaped Arthropod Handling	Escaped arthropods are killed or collected and disposed of.	Loose arthropods must be killed and disposed of, or recaptured and returned to the container from which they escaped.  Infected arthropods must not be killed with bare hands, and must be transferred using filtered mechanical or vacuum aspirators if infected.	Loose arthropods must be killed and disposed of, or recaptured and returned to the container from which they escaped.  Infected arthropods are not killed with hands, and must be transferred using filtered mechanical or vacuum aspirators.   Only personnel properly trained and equipped to work with designated arthropods and BSL-3 infectious agents are to recover and/or kill escaped arthropods.
Accidental Release Reporting	The insectary director is notified promptly of accidental release of vectors.	A release procedure is developed and posted.  This includes contacts and immediate mitigating actions.  Accidents that result in release of infected arthropods from primary containment vessels, or that result in overt exposure to infectious material must be reported immediately to the insectary director who is responsible for ensuring that appropriate and documented action is taken to mitigate the release. Location, number, and type of material is prominently posted until the source is eliminated.  Followup medical evaluation, surveillance, and treatment are provided as appropriate, and written records are maintained.	ACL-2
Movement of Equipment		All equipment must be appropriately decontaminated and disinfested prior to transfer between rooms within the insectary, and prior to removal from the insectary.	ACL-2
Inventory of Arthropods			In addition to appropriate primary containment cages, when possible, the number of arthropods must be included on the label, and records are maintained to account for all arthropods from the time of transfer to the ACL-3 insectary to the time of termination. Vessels containing low mobility stages (e.g., eggs, pupae, hibernating adults) should not be stored within the ACL-3 insectary unless they meet the ACL-3 criteria.
		Safety Equipment
Eye and Face Protection		Appropriate face/eye and respiratory protection is worn by all personnel entering the insectary.	ACL-2
Gloves	Gloves are worn when handling host animals or blood used to feed the arthropods.	Gloves are worn when handling potentially infected arthropods, blood, and associated equipment and when contact with potentially infectious material is unavoidable.	Personnel wear gloves when handling infected arthropods or host animals and associated equipment. Gloves are removed aseptically.
Torso Apparel	White laboratory coats, gowns, and/or uniforms are worn at all times in the insectary when handling blood and vertebrate animals.	White laboratory coats, gowns, and/or uniforms are worn at all times in the insectary when handling blood, vertebrate animals, and infected materials.	White laboratory coats, gowns, and/or uniforms in the insectary are worn at all times by all personnel entering the insectary.  Wrap-around or solid-front gowns are worn over this clothing. Front-button laboratory coats alone are unsuitable. The gowns are removed and left in the insectary.  Before leaving the insectary, scrub suits and uniforms are removed and appropriately contained and decontaminated prior to laundering or disposal.
Foot Apparel			Boot, shoe covers, or other protective footwear, and disinfectant foot baths (with appropriate anti-arthropod measures) are available and used where indicated.
Personal Clothing		Clothing should minimize the area of exposed skin (e.g., skirts, shorts, open toed shoes, sandals, tee shirts are inadvisable), since this can increase the risk of attracting and being bitten by a loose arthropod.	ACL-2
Arthropod-specific Personal Protective Equipment	Personal protective equipment is worn as appropriate e.g., respirators for arthropod-associated allergies, particle masks, head covers.	In addition to ACL-1 measures, personal protection equipment is used for all activities involving manipulations of infected or potentially infected arthropods.	ACL-2
Pesticide			Pesticide for emergency use is available in areas in which escape of arthropods is likely.
		Facilities
Location of Insectary	The insectary area is separated from areas that are used for general traffic within the building.	The insectary is separated from areas that are open to unrestricted personnel traffic within the building.  It is recommended that this be accomplished by at least two self-closing doors that prevent passage of the arthropods.  Increased levels of physical isolation are recommended e.g. separate buildings, wings, suites etc.	The insectary is strictly separated from areas that are open to unauthorized, untrained personnel within the building by locked doors.  These are opened by key lock, proximity reader, card key, etc.
Insectary Doors	Doors openings, whether covered by rigid panels , glass, screens, plastic sheets or cloth, minimize escape and entrance of arthropods.	Recommended entrance to the insectary is via a double-door vestibule that prevents flying and crawling arthropod escape.  For example, the two contiguous doors must not be opened simultaneously. Internal doors may open outwards or be sliding, but are self-closing, and are kept closed when arthropods are present.  Additional barriers (e.g., hanging curtains) are highly recommended.	Access to the facility is limited to trained, approved personnel by a self-closing and self-locking door.  The external insectary entry doors are controlled by a key lock, card key, or proximity reader.  Entry into the insectary is via a double-door entry that includes a change room and shower(s).  Showers are plumbed to prevent arthropod escape.  An additional double-door access (air lock) or double-door autoclave may be provided for movement of supplies and wastes into and out of the facility respectively.  The two contiguous doors must never be opened simultaneously. Internal doors may open outwards or be sliding, but are self-closing, and are kept closed when arthropods are present.  Additional barriers (e.g., hanging curtains) are recommended.
Insectary Windows	Windows, if present, effectively prevent escape of the smallest arthropods present.	Windows are not recommended, but if present cannot be opened and are well-sealed.  Windows must be resistant to breakage (e.g., double paned or wire-reinforced).	Windows are not recommended. Any windows present are resistant to breakage (e.g. double paned or wire-reinforced), and well sealed.  If present, fixed light windows are recommended.
Vacuum Systems		If a central vacuum system is installed, each service outlet is fitted with suitable barriers/filters to prevent arthropod escape.  Filters are installed to permit decontamination and servicing.   Other vacuum devices are appropriately filtered to prevent transfer and exhausting of arthropods.	ACL-2
Interior Surfaces		The insectary is designed, constructed and maintained to facilitate cleaning and housekeeping. The interior walls are light-colored so that a loose arthropod can be easily located, recaptured or killed. Gloss finishes, ideally resistant to chemical disinfectants and fumigants, are recommended. Floors are light colored, smooth and uncovered. Ceilings are as low as possible to simplify detection and capture of flying insects.	In addition to the recommendations for ACL-2, spaces around doors are sealed to facilitate decontamination or troughs surrounding door-frames can be installed and filled with sticky or greasy material that will trap crawling arthropods.
Floor Drains		Floor drains are modified to prevent accidental release of arthropods and agents.  If present, traps must be filled with an appropriate chemical treatment to prevent survival of all arthropod stages (e.g. mosquito larvae).	Floor drains are not recommended.  If present, traps must be filled with an appropriate treatment to prevent survival of any arthropod stage (e.g. mosquito larvae). Ideally, all drains are plumbed to a holding tank to facilitate heat or chemical treatment to kill all stages of arthropod prior to disposal into the waste system.
Plumbing and Electrical Fixtures		Internal facility appurtenances (e.g., light fixtures, pipes, ducting) are minimal since these provide hiding places for loose arthropods.  Penetrations of walls, floors and ceilings are minimal and sealed/caulked.  Ideally, light fixtures are flush with the ceiling, sealed and accessed from above.	ACL-2
HVAC		Ventilation is appropriate for arthropod maintenance, but does not compromise containment of the agent or arthropod.  Examples include: exhaust air is discharged to the outside without being recirculated to other rooms.  Appropriate filter/barriers are installed to prevent escape of arthropods. The direction of airflow in the insectary is inward. A progressively negative pressure gradient is maintained as distance from the main entrance increases. Fans located in the vestibule and internal corridor can be used to help prevent escape of flying arthropods. Air curtains are located in vestibules and doorways.	Ventilation is appropriate for arthropod maintenance, but does not compromise containment.  Exhaust air is discharged to the outside without being re-circulated to other rooms.  Exhaust must be dispersed away from occupied areas and air intakes, or the exhaust must be HEPA-filtered. Appropriate filter/barriers are installed to prevent escape of arthropods. The direction of airflow in the insectary is inward. A progressively negative pressure gradient is maintained as distance from the main entrance increases. Personnel must verify that the direction of the airflow is proper (a visual monitoring device/meter is recommended to confirm directional inward airflow).  Audible alarms alert personnel to system failure.
Sterilization Equipment		An autoclave is available conveniently located to rooms containing arthropods within the insectary building. .	An autoclave is available within the suite of rooms containing arthropods.
Sink and Shower		The facility has a hand-washing sink with hot water and with suitable plumbing to prevent arthropod escape.	In addition to the ACL-2 recommendation, an appropriately plumbed shower is available within the insectary suite.
Illumination		Illumination is appropriate for arthropod maintenance but does not compromise arthropod containment, impede vision or adversely influence the safety of procedures within the insectary.  Lighted (or dark) openings that attract escaped arthropods are avoided.	ACL-2
BSC			HEPA-fitted exhaust air from Class II biological safety cabinets can be re-circulated into the insectary provided that it is certified annually. If exhausting to the outside, the cabinet must be installed appropriately. If Class III cabinets are used they must be installed appropriately.
Facility Compliance Monitoring		The facility is evaluated annually for compliance to the ACL-2 level.  The principle investigator or insectary director inspects the facility annually to ensure that alterations and maintenance have not compromised the containment characteristics.  Adequacy of the practices and facility in view of changes in research protocols, agents, or arthropods are considered.	The completed ACL-3 insectary design and operational procedures must be documented.  The insectary must be tested for verification that the design and operational parameters have been met prior to operation. ACL-3 insectaries are re-verified at least annually against these procedures as modified by operational experience.