APPENDIX A

Biological Safety Cabinets

	Biological Safety Cabinets (BSCs) are among the most effective, as well as the most commonly used primary containment devices in laboratories working with infectious agents.  The three general types available (Class I, II, III) have performance characteristics and applications which are described in this appendix.

	Properly maintained Class I and II BSCs, when used in conjunction with good microbiological techniques, provide an effective containment system for safe manipulation of moderate and high-risk microorganisms (Biosafety Level 2 and 3 agents).  Both Class I and II BSCs have inward face velocities (75-100 linear feet per minute) that provide comparable levels of containment for laboratory workers and the immediate environment from infectious aerosols generated within the cabinet.  Class II BSCs have the additional advantage of providing protection to the research material by high-efficiency particulate air (HEPA)-filtration of the air flow down across the work surface (vertical laminar flow).  Class III cabinets offer the maximum protection to laboratory personnel, the community, and the environment because all hazardous materials are contained in a totally enclosed, ventilated cabinet.

CLASS I

(Note: Class I BSCs are no longer being manufactured on a regular basis; many have been replaced by Class II BSCs.)	

	The Class I Biological Safety Cabinet (Fig. 1) is a negative-pressure, ventilated cabinet usually operated with an open front and a minimum face velocity at the work opening of at least 75 linear feet per minute (lfpm).  All of the air from the cabinet is exhausted through a HEPA filter either into the laboratory, or to the outside.  The Class I BSC is designed for general microbiological research with low and moderate risk agents, and is useful for containment of mixers, blenders, and other equipment.  These cabinets are not appropriate for handling research materials that are vulnerable to airborne contamination, since the inward flow of unfiltered air from the laboratory can carry microbial contaminants into the cabinet.

	The Class I BSC can also be used with an installed front closure panel without gloves that will increase the inward flow velocity to approximately 150 lfpm.  If such equipped cabinets are ducted to the outside exhaust, they may be used for toxic or radiolabelled materials used as an adjunct to microbiological research.  Additionally, arm-length rubber gloves may be attached to the front panel with an inlet air pressure release for further protection.

CLASS II

	The Class II Biological Safety Cabinet (Fig. 2) is designed with inward air flow at a velocity to protect personnel (75-100 lfpm), HEPA-filtered vertical laminar airflow for product protection, and HEPA-filtered exhaust air for environmental protection.  Design, construction and performance standards for Class II BSCs, as well as a list of products that meet these standards, have been developed by and are available from the National Sanitation Foundation International,136 Ann Arbor, Michigan.  Utilization of this standard and list should be the first step in selection and procurement of a Class II BSC.

	Class II BSCs are classified into two types (A and B) based on construction, air flow velocities and patterns, and exhaust systems. Basically, Type A cabinets are suitable for work with microbiological research in the absence of volatile or toxic chemicals and radionuclides, since air is recirculated within the work area.  Type A cabinets may be exhausted through HEPA filters into the laboratory, or to the outside via a "thimble" connection to the exhaust ductwork.

	Type B cabinets are further sub-typed into types B1, B2, and B3.  A comparison of the design features and applications are found in Table 1 of this appendix.  Type B cabinets are hard-ducted to the exhaust system, and contain negative pressure plena.  These features, plus an increased face velocity of 100 lfpm, allow work to be done with toxic chemicals or radionuclides.

	It is imperative that Class I and II biological safety cabinets are tested and certified in situ at the time of installation within the laboratory, at any time the BSC is moved, and at least annually thereafter.  Certification at locations other than the final site may attest to the performance capability of the individual cabinet or model but does not supersede the critical certification prior to use in the laboratory.

	As with any other piece of laboratory equipment, personnel must be trained in the proper use of the biological safety cabinets.  Of particular note are those activities which may disrupt the inward directional airflow through the work opening of Class I and II cabinets.  Repeated insertion and withdrawal of the workers' arms in and from the work chamber, opening and closing doors to the laboratory or isolation cubicle, improper placement or operation of materials or equipment within the work chamber, or brisk walking past the BSC while it is in use are demonstrated causes of the escape of aerosolized particles from within the cabinet.  Class I and II cabinets should be located away from traffic patterns and doors.  Fans, heating and air conditioning registers, and other air handling devices can also disrupt airflow patterns if located adjacent to the BSC.  Strict adherence to recommended practices for the use of BSCs and proper placement in the laboratory are important in attaining the maximum containment capability of the equipment as is the mechanical performance of the equipment itself.

CLASS III

	The Class III Biological Safety Cabinet (Fig. 3) is a totally enclosed, ventilated cabinet of gas-tight construction and offers the highest degree of personnel and environmental protection from infectious aerosols, as well as protection of research materials from microbiological contaminants.  Class III cabinets are most suitable for work with hazardous agents that require Biosafety Level 3 or 4 containment.

	All operations in the work area of the cabinet are performed through attached rubber gloves.  The Class III cabinet is operated under negative pressure.  Supply air is HEPA-filtered, and the cabinet exhaust air is filtered by two HEPA filters in series, or HEPA filtration followed by incineration, before discharge outside of the facility.

	All equipment required by the laboratory activity, such as incubators, refrigerators, and centrifuges, must be an integral part of the cabinet system,  The Class III cabinet must be connected to double-doored autoclaves and chemical dunk tanks to sterilize or disinfect all materials exiting the cabinet, and to allow supplies to enter the cabinet.  Several Class III cabinets are therefore typically set up as an interconnected system.

POSITIVE-PRESSURE PERSONNEL SUIT

	Personnel protection equivalent to that provided by Class III cabinets can also be obtained with the use of a one-piece, ventilated suit for the laboratory worker when working with Biosafety Level 3 or 4 agents in a "suit area" and using Class I or II BSCs.  The personnel suit is maintained under positive pressure with a life support system to prevent leakage into the suit.  In this containment system, the worker is isolated from the work materials.

	The personnel suit area must be essentially equivalent to a large Class III cabinet.  The area is entered through an air-lock fitted with airtight doors.  A chemical shower is provided as a "dunk tank" to decontaminate the surfaces of the suit as the worker leaves the area.  The exhaust air from the suit area is filtered by two HEPA filter units installed in series.  The entire area must be under negative pressure.

	As in the case with Class III BSCs, the gloves of the personnel suit are the most vulnerable component of the system, as they are subject to punctures by sharps or animal bites. 
	
	(Caution:  Horizontal laminar flow "clean benches" are used in clinical, pharmaceutical, and laboratory facilities strictly for product protection.  Since the worker sits in the immediate downstream exhaust from the "clean bench", this equipment must never be used for handling toxic, infectious, radioactive, or sensitizing materials.)

Figure 1.  Class I Biological Safety Cabinet.  A. front opening, B. work surface, C. window, D. exhaust plenum, E. HEPA filter.

Figure 2(a).  Class II, Type A BSC.  A. blower, B. rear plenum, C. supply HEPA filter, D. exhaust, E. sash, F. work surface.

Figure 2(b).  Class II, Type B1 BSC.  A. blowers, B. supply HEPA filters, C. sliding sash, D. positive pressure plenums, E. additional supply HEPA filter or back-pressure plate, F. exhaust HEPA filter, G. negative pressure exhaust plenum, H. work surface

Figure 2(c).	Class II, Type B2 BSC.  A. storage cabinet, B. work surface, C. sliding sash, D. lights, E. supply HEPA filter, F. exhaust HEPA filter, G. supply blower, H. control panel, I. filter screen, J. negative pressure plenum.

Figure 2(d).	Table-top model of a Class II, Type B3 BSC.  A. front opening, B. sliding sash, C. light, D. supply HEPA filter, E. positive pressure plenum, F. exhaust HEPA filter, G. control panel, H. negative pressure plenum, I. work surface.

Figure 3.	Class III BSC.  A. stand, B. glove ports, C. O-ring for attaching arm-length gloves to cabinet, D. slopped glass viewing window, E. supply HEPA filter, F. exhaust HEPA filter (Note that the second exhaust HEPA filter required for Class III cabinets is not depicted in this diagram), G. double-ended autoclave.

APPENDIX B

Immunoprophylaxis

	An additional level of protection for at-risk personnel may be achieved with appropriate prophylactic immunizations.  A written organizational policy which defines at-risk personnel, which specifies risks as well as benefits of specific vaccines, and which distinguishes between required and recommended vaccines is essential.  In developing such an organizational policy, these recommendations and requirements should be specifically targeted at infectious diseases known or likely to be encountered in a particular facility.

	Vaccines for which the benefits (levels of antibody considered to be protective) clearly exceed the risks (local or systemic reactions) should be required for all clearly identified at-risk personnel.  Examples of such preparations include vaccines against hepatitis B, yellow fever, rabies, and poliomyelitis.  Recommendations for giving less efficacious vaccines, those associated with high rates of local or systemic reactions, or those that produce increasingly severe reactions with repeated use should be carefully considered.  Products with these characteristics (e.g., cholera, tularemia, and typhoid vaccines) may be recommended but should not ordinarily be required for employment.  A complete record of vaccines received on the basis of occupational requirements or recommendations should be maintained in the employee's permanent medical file.

	Recommendations for the use of vaccines, adapted from those of Public Health Service Advisory Committee on Immunization Practices, are included in the agent summary statements in Section VII.  Particular attention must be given to individuals who are or may become immunocompromised, as recommendations for vaccine administration may be different than for immunologically competent adults.

APPENDIX C

Surveillance of Personnel for Laboratory-Associated Rickettsial Infections

	Under natural circumstances, the severity of disease caused by rickettsial agents varies considerably.  In the laboratory, very large inocula which might produce unusual and perhaps very serious responses are possible.  Surveillance of personnel for laboratory-associated infections with rickettsial agents can dramatically reduce the risk of serious consequences of disease.
	
	Experience indicates that infections treated adequately with specific anti-rickettsial chemotherapy on the first day of disease do not generally present serious problems.  Delay in instituting appropriate chemotherapy, however, may result in debilitating or severe acute disease ranging from increased periods of convalescence in typhus and scrub typhus to death in R. rickettsii infections.  The key to reducing the severity of disease from laboratory-associated infections is a reliable surveillance system which includes (1) round-the-clock availability of an experienced medical officer, (2) indoctrination of all personnel into the potential hazards of working with rickettsial agents and advantages of early therapy, (3) a reporting system for all recognized overt exposures and accidents, (4) the reporting of all febrile illnesses, especially those associated with headache, malaise, prostration, when no other certain cause exists and, (5) a non-punitive atmosphere that encourages reporting of any febrile illness.

	Rickettsial agents can be handled in the laboratory with minimal real danger to life when an adequate surveillance system complements a staff who are knowledgeable about the hazards of rickettsial infections and who put to use the safeguards recommended in the agent summary statements.

APPENDIX D

Importation and Interstate Shipment of Human Pathogens and Related Materials

	The importation or subsequent receipt of etiologic agents and vectors of human disease is subject to the Public Health Service Foreign Quarantine Regulations (42 CFR, Section 71.156).  Permits authorizing the importation or receipt of regulated materials and specifying conditions under which the agent or vector is shipped, handled, and used are issued by the Centers for Disease Control and Prevention.

	The interstate shipment of indigenous etiologic agents, diagnostic specimens, and biologic products is subject to applicable packaging, labeling, and shipping requirements of the Interstate Shipment of Etiologic Agents (42 CFR Part 72).  Packaging and labeling requirements for interstate shipment of etiologic agents are summarized and illustrated in Figure 4.

	Persons needing to report leaking damaged packages of etiologic agents may call 1-800-232-0124.

	Additional information on the importation and interstate shipment of etiologic agents of human disease, diagnostic specimens, and other related materials may be obtained by contacting:

Centers for Disease Control and Prevention
Attention: Biosafety Branch
Office of Health and Safety, Mail Stop F-05
1600 Clifton Road N.E.
Atlanta, Georgia  30333
Telephone: (404) 639-3883
Fax: (404) 639-2294

Figure 4.  Packing and Labeling of Etiologic Agents

	Figures 4a and 4b diagram the packaging and labelling of etiologic agents in volumes of less than 50 ml. in accordance with the provisions of subparagraph 72.3(a) of the regulation on Interstate Shipment of Etiologic Agents (42 CFR, Part 72). A revision has been proposed that may result in additional package labeling requirements, but this has not been issued as of the publication of this third edition of BMBL.

For further information on any provision of this regulation contact:
			Centers for Disease Control and Prevention
			Attn: Biosafety Branch Chief
			Mail Stop F-05
			1600 Clifton Road N.E.
			Atlanta, GA  30333
			Telephone: (404) 639-3883
			FAX: (404) 639-2294

Note that the shipper's name, address and telephone number must be on the outer and inner containers.  The reader is also advised to refer to additional provisions of the Department of Transportation (49 CFR, Parts 171-180) Hazardous Materials Regulations.
APPENDIX E

Restricted Animal Pathogens

	Nonindigenous pathogens of domestic livestock and poultry may require special laboratory design, operation, and containment features not generally addressed in this publication.  The importation, possession, or use of the following agents is prohibited or restricted by law or by U.S. Department of Agriculture regulations or administrative policies:

African horse sickness African Swine fever virus
Akabane virus
Besnoitia besnoiti
Borna disease virus
Bovine spongiform en-          cephalopathy
Bovine infectious petechial     fever agent
Brucellosis melitensis
Camelpox virus
Cochliomyia hominivorax      (screwworn)
Ephermeral fever virus
Foot and mouth disease        virus
Fowl plague virus (lethal       avian influenza)
Hog cholera virus
Histoplasma (Zymonema)      farciminosum
Louping ill virus
Lumpy skin disease virus
Mycoplasma agalactiae
Mycoplasma mycoides
Nairobi sheep disease  virus (Ganjam      virus)
Newcastle disease virus                        (velogenic strains)
Peste des petits ruminants
  (pest of small ruminants)
Pseudomonas ruminantium
  (heartwater)
Rift Valley fever virus
Riderspest virus
Sheep ant goat pox
Swine vesicular disease virus
Teschen disease virus
Theileria annulata
Theileria lawrencei
Theileria bovis
Theileria hirci
Trypanosoma evansi
Trypanosoma vivax
Vesicular exanthema virus
Viral hemorrhagic disease of rabbits
Wesselsbron disease virus

	The importation, possession, use, or interstate shipment of animal pathogens other than those listed above may also be subject to regulations of the U.S. Department of Agriculture.

	Additional information may be obtained by writing to:

	U.S. Department of Agriculture
	Animal and Plant Health Inspection Service
	Veterinary Services, Import-Export Products Staff
	Room 756, Federal Building
	6505 Belcrest Road 
	Hyattsville, Maryland  20782
	Telephone:	(301) 436-7830 or
			(301) 436-8499
	Fax:		(301) 436-8226 

APPENDIX F

Resources for Information

	Resources for information, consultation, and advice on biohazard control, decontamination procedures, and other aspects of laboratory safety management include:

Centers for Disease Control and Prevention
Attention:  Biosafety Branch
Atlanta, Georgia  30333
Telephone:  (404) 329-3883

National Institutes of Health  
Attention:  Division of Safety
Bethesda, Maryland  20205
Telephone:  (301) 496-1357

National Animal Disease Center
U.S. Department of Agriculture
Ames, Iowa  50010
Telephone:  (515) 862-8258