Return to Chapter 26 Biosafety.
DRAFT
Contents
Appendix A. Biological Safety Worker Health & Safety Program (10 CFR 851)
Appendix B. Pathogen and Toxin Lists
Appendix C. Laboratory Biosafety Level 1 and Biosafety Level 2 Criteria
Appendix D. Good Microbiological Practice
Appendix E Biosafety Cabinets
Appendix F. Decontamination and Antimicrobials
Appendix G. Biological Spills and Cleanup
Appendix H. Transportation and Shipping
Appendix I. Import, Export, and Transfer Restrictions
Appendix A. Biological Safety Worker Health & Safety Program (10 CFR 851)
A.1 Scope
This appendix describes specific requirements and program elements for biological etiologic agents and select agents that are included in the Lawrence Berkeley National Laboratory (Berkeley Lab or LBNL) Worker Health and Safety Program and Biosafety Program, as required by the DOE Worker Safety and Health Program (10 CFR 851, Appendix A, Section 7).
A.2 General Policy
Work with biological etiologic agents and any select agents at Berkeley Lab is conducted in a safe, environmentally sound, and compliant manner using the principles and functions of Integrated Safety Management (ISM) and work authorization. Line managers and researchers define their work with biological materials, evaluate the biological hazards, determine the risk, and implement required biosafety containment controls (e.g., establish a biosafety level). This is accomplished with the assistance and oversight of the Institutional Biosafety Committee (IBC), the Environment/Health/Safety (EHS) Division (e.g., Health & Safety, Waste Management, and Health Services), and other Berkeley Lab ES&H functions as part of the Biosafety Program.
A.3 Definitions
Biological etiologic agents and select agents have the following specific definitions.
- Biological Etiologic Agents are agents of biological origin (e.g., bacterium, fungus, parasite, virus, etc.) that cause disease in humans (i.e., are pathogenic to humans). See Appendix B of the Biosafety Program or the NIH Guidelines for a list and risk group categorization of human etiologic agents. Agents requiring implementation of Biosafety Level 3 or 4 containment are not used or stored at Berkeley Lab
- Select Agents. The term “select agents” is used to describe a list of specific pathogenic agents that are strictly regulated by the Centers for Disease Control and Prevention (CDC) and U.S. Department of Agriculture (USDA) because they may be used as biological weapons or pose a severe threat to human, animal, and plant health. See Appendix B of the Biosafety Program or the National Select Agent Registry for a list of select agents.
A.4 Program Elements
LBNL’s Biosafety Program for managing biological etiologic agents and select agents under the LBNL Worker Health and Safety Program Plan includes the program elements listed below:
- LBNL’s Institutional Biosafety Committee (IBC) reviews the following work, plans, and procedures.
- Work Review and Assessment. The IBC (i) reviews work with biological etiologic agents for compliance with applicable CDC, NIH, WHO, and other international, federal, state, and local guidelines applicable to biological etiologic agents, and (ii) assesses the containment level, facilities, procedures, practices, training, and expertise of personnel using these agents. Applicable guidelines include the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules, the CDC-NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), and the WHO Laboratory Biosafety Manual.
- Security, Safeguards, and Emergency Management Plan and Procedure Review. The IBC reviews LBNL’s security, safeguards, and emergency management plans and procedures to ensure they adequately consider work involving biological etiologic agents.
- Agent Inventory and Status Report. The EHS Division Health & Safety Research Support Team maintains an inventory and status of biological etiologic agents, and provides the DOE Berkeley Site Office (through the LBNL IBC) with an annual status report describing the status and inventory of biological etiologic agents and their Biosafety Program. The inventory includes a list of agents, the principal investigator’s or owner’s name, and location(s) of the researcher’s laboratory operation.
- Select Agent Application for Registration. Berkeley Lab did not possess or have plans to possess select agents when this Appendix was last revised. If Berkeley Lab researchers request use of a select agent, the requirements related to select agents in this Appendix will be implemented. The LBNL EHS Division Responsible Official (RO) or Alternate Responsible Official (ARO) for select agents submits any and each CDC Laboratory Registration or Select Agent Program registration application package to the U.S. Department of Energy (DOE) Berkeley Site Office (BSO) when the application is registering a new laboratory facility for the purpose of transferring, receiving, or handling select agents. DOE’s review of the application and concurrence is required before transmittal to the CDC.
- Select Agent Transfer, Disposition, and/or Destruction. The LBNL EHS RO or ARO for select agents submits to the DOE BSO:
- A copy of each CDC Transfer of Select Agents form (i) upon initial submission of the form to a vendor or other supplier requesting or ordering a select agent for transfer, receipt, and handling in a registered facility, and (ii) within 10 days upon completion of the form documenting final disposition of the select agent.
- Documentation of the destruction of a complete stock of a select agent within 10 days of the destruction.
- Safeguards, Security, and Emergency Management Plans and/or Program Confirmation. The Protective Services Department’s Security and Emergency Services and the EHS Health & Safety Research Support Team confirm that the site safeguards and security plans and emergency management programs address biological etiologic agents and any biological select agents.
- Immunization Policy. The IBC (which includes the LBNL Medical Director and Biosafety Officer) assesses potential vaccines and the need for immunizations when it reviews work to be conducted with biological etiologic agents. Any immunization recommendations are then incorporated into the research operation’s biosafety documentation.
____________________
Appendix B. Pathogen and Toxin Lists
B.1 Introduction and Scope
Pathogens and toxins are discussed in detail in Work Process B.3.d, Pathogenic Agents and Toxins, of this manual. This appendix lists the following biological agents and toxins presented in Work Process B.3.d:
- Human etiologic agents (pathogens) from Appendix B of the NIH Guidelines
- Select agents and toxins from the National Select Agent Registry (NSAR)
- Plant pathogens previously identified by U.S. Department of Agriculture (USDA)
These lists are provided for convenience in this manual, but may not reflect the actual regulatory list or applicable agents or materials. Regulatory sources, standards, and Web links noted in this appendix and Work Process B.3.d should be consulted to confirm applicable agents or toxins.
B.2 NIH Guidelines Human Etiologic Agents
This section provides a list of human pathogens and their Risk Group (RG) 2, RG3, and RG4 designations as excerpted from Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, of the NIH Guidelines, amendment effective November 6, 2013.
B.2.1 Risk Group 1 Agents
RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis (see NIH Guidelines, Appendix C-IV-A, Bacillus subtilis or Bacillus licheniformis Host-Vector Systems, Exceptions); adeno-associated virus (AAV, all serotypes); and recombinant or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and which are produced in the absence of a helper virus. A strain of Escherichia coli (see NIH Guidelines, Appendix C-II-A, Escherichia coli K-12 Host Vector Systems, Exceptions) is an RG1 agent if it (a) does not possess a complete lipopolysaccharide (i.e., lacks the O antigen) and (b) does not carry any active virulence factors (e.g., toxins) or colonization factors and does not carry any genes encoding these factors.
Those agents not listed in RGs 2, 3, and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the unlisted agents and their relationship to the listed agents.
B.2.2 Risk Group 2 Agents
RG2 agents are associated with human disease that is rarely serious and for which preventive or therapeutic interventions are often available.
Risk Group 2 Bacterial Agents, Including Chlamydia
- Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)
- Actinobacillus
- Actinomyces pyogenes (formerly Corynebacterium pyogenes)
- Aeromonas hydrophila
- Amycolata autotrophica
- Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum)
- Arizona hinshawii: all serotypes
- Bacillus anthracis
- Bartonella henselae, B. quintana, B. vinsonii
- Bordetella including B. pertussis
- Borrelia recurrentis, B. burgdorferi
- Burkholderia (formerly Pseudomonas species) (except those listed in NIH Guidelines, Appendix B-III-A (RG3))
- Campylobacter coli, C. fetus, C. jejuni
- Chlamydia psittaci, C. trachomatis, C. pneumoniae
- Clostridium botulinum, C. chauvoei, C. haemolyticum, C. histolyticum, C. novyi, C. septicum, C. tetani
- Coxiella burnetii,specifically the Phase II,Nine Mile strain, plaque purified, clone 4
- Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale
- Dermatophilus congolensis
- Edwardsiella tarda
- Erysipelothrix rhusiopathiae
- Escherichia coli: all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen, including E. coli O157:H7
- *Francisella tularensis, specifically*F. tularensis subspecies novicida (aka F. novicida), strain Utah 112;*F.tularensis subspecies holarctica LVS;*F tularensis biovar tularensis strain ATCC 6223 (aka strain B38). *For research involving high concentrations, BL3 practices should be considered.
- Haemophilus ducreyi, H. influenzae
- Helicobacter pylori
- Klebsiella: all species except K. oxytoca (RG1)
- Legionella, including L. pneumophila
- Leptospira interrogans: all serotypes
- Listeria
- Moraxella
- Mycobacterium (except those listed in Appendix B-III-A [RG3]), including M. avium complex, M. asiaticum, M.bovis BCG vaccine strain, M. chelonei, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M. marinum, M.paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi
- Mycoplasma, except M. mycoides and M. agalactiae, which are restricted animal pathogens
- Neisseria gonorrhoeae, N. meningitidis
- Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. Transvalensis
- Pseudomonas aeruginosa
- Rhodococcus equi
- Salmonella, including S. arizonae, S. cholerasuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S.paratyphi, A, B, C, S. typhi, S. typhimurium
- Shigella, including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei
- Sphaerophorus necrophorus
- Staphylococcus aureus
- Streptobacillus moniliformis
- Streptococcus, including S. pneumoniae, S. pyogenes
- Treponema pallidum, T. carateum
- Vibrio cholerae, V. parahemolyticus, V. vulnificus
- Yersinia enterocolitica
- Yersinia pestis,specificallypgm(–)strains (lacking the 102 kb pigmentation locus) andlcr(–)strains (lacking the LCR plasmid)
Risk Group 2 Fungal Agents
- Blastomyces dermatitidis
- Cladosporium bantianum, C. (Xylohypha) trichoides
- Cryptococcus neoformans
- Dactylaria galopava (Ochroconis gallopavum)
- Epidermophyton
- Exophiala (Wangiella) dermatitidis
- Fonsecaea pedrosoi
- Microsporum
- Paracoccidioides braziliensis
- Penicillium marneffei
- Sporothrix schenckii
- Trichophyton
Risk Group 2 Parasitic Agents
- Ancylostoma human hookworms, including A. duodenale, A. ceylanicum
- Ascaris, including Ascaris lumbricoides suum
- Babesia, including B. divergens, B. microti
- Brugia filaria worms, including B. malayi, B. timori
- Coccidia
- Cryptosporidium, including C. parvum
- Cysticercus cellulosae (hydatid cyst, larva of T. solium)
- Echinococcus, including E. granulosis, E. multilocularis, E. vogeli
- Entamoeba histolytica
- Enterobius
- Fasciola, including F. gigantica, F. hepatica
- Giardia, including G. lamblia
- Heterophyes
- Hymenolepis, including H. diminuta, H. nana
- Isospora
- Leishmania, including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruvania, L. tropica
- Loa loa filaria worms
- Microsporidium
- Naegleria fowleri
- Necator human hookworms, including N. americanus
- Onchocerca filaria worms, including O. volvulus
- Plasmodium, including simian species, P. cynomologi, P. falciparum, P. malariae, P. ovale, P. vivax
- Sarcocystis, including S. sui hominis
- Schistosoma, including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi
- Strongyloides, including S. stercoralis
- Taenia solium
- Toxocara, including T. canis
- Toxoplasma, including T. gondii
- Trichinella spiralis
- Trypanosoma, including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi
- Wuchereria bancrofti filaria worms
Risk Group 2 Viruses
- Adenoviruses, human: All types
- Alphaviruses (Togaviruses), group A Arboviruses:
- Chikungunya vaccine strain 181/25
- Eastern equine encephalomyelitis virus
- Venezuelan equine encephalomyelitis vaccine strain TC-83
- Western equine encephalomyelitis virus
Arenaviruses:
- Junin virus candid #1 vaccine strain
- Lymphocytic choriomeningitis virus (non-neurotropic strains)
- Tacaribe virus complex
- Other viruses as listed in BMBL
- Bunyaviruses:
- Bunyamwera virus
- Rift Valley fever virus vaccine strain MP-12\
- Other viruses as listed in BMBL
- Calciviruses
- Coronaviruses
- Flaviviruses, Group B Arboviruses:
- Dengue virus, serotypes 1, 2, 3, and 4
- Japanese encephalitis virus (except those strains listed in NIH Guidelines Appendix B-II-D Risk Group 2 (RG2) – Viruses)
- Yellow fever virus vaccine strain 17D
- Other viruses as listed in BMBL
- Hepatitis A, B, C, D, and E viruses
- Herpesviruses, except Herpesvirus simiae (monkey B virus) (see Appendix B-IV-D, Risk Group 4 (RG4) – Viral Agents):
- Cytomegalovirus
- Epstein Barr virus
- Herpes simplex, types 1 and 2
- Herpes zoster
- Human herpesvirus, types 6 and 7
- Orthomyxoviruses:
- Influenza viruses, types A, B, and C
- Tick-borne orthomyxoviruses
- Papilloma viruses: All human papilloma viruses
- Paramyxoviruses:
- Newcastle disease virus
- Measles virus
- Mumps virus
- Parainfluenza viruses, types 1, 2, 3, and 4
- Respiratory syncytial virus
- Parvoviruses: Human parvovirus (B19)
- Picornaviruses:
- Coxsackie viruses, types A and B
- Echoviruses, all types
- Polioviruses, all types, wild and attenuated
- Rhinoviruses, all types
- Poxviruses: All types except monkeypox virus (see Appendix B-III-D, Risk Group 3 (RG3)—Viruses and Prions) and restricted poxviruses including Alastrim, Smallpox, and Whitepox (see BMBL Section V-L)
- Reoviruses: All types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus)
- Rhabdoviruses
- Rabies virus, all strains
- Vesicular stomatitis virus non exotic strains: VSV-Indiana 1 serotype strains (e.g.,Glasgow, Mudd-Summers, Orsay, San Juan) and VSV-New Jersey serotype strains (e.g.,Ogden, Hazelhurst)
- Rubivirus (Togaviruses), Rubella virus
B.2.3 Risk Group 3 Agents
RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.
Risk Group 3 Bacterial Agents Including Rickettsia
- Bartonella
- Brucella including B. abortus, B. canis, B. suis
- Burkholderia (Pseudomonas) mallei, B. pseudomallei
- Coxiella burnetii (except the Phase II,Nine Mile strain listed in NIH Guidelines Appendix B-II-A, Risk Group 2 (RG2) – Bacterial Agents Including Chlamydia)
- Francisella tularensis (except those strains listed in NIH Guidelines Appendix B-II-A, Risk Group 2 (RG2) – Bacterial Agents Including Chlamydia)
- Mycobacterium bovis (except BCG strain, see NIH Guidelines Appendix B-II-A, Risk Group 2 (RG2) – Bacterial Agents
- Including Chlamydia), M. tuberculosis
- Pasteurella multocida type B: “Buffalo” and other virulent strains
- Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R. tsutsugamushi, R. typhi (R. mooseri)
- Yersinia pestis (except those strains listed in NIH Guidelines Appendix B-II-A, Risk Group 2 (RG2) – Bacterial Agents Including Chlamydia)
Risk Group 3 Fungal Agents
- Coccidioides immitis (sporulating cultures, contaminated soil)
- Histoplasma capsulatum, H. capsulatum var. duboisii
Risk Group 3 Parasitic Agents
None
Risk Group 3 Viruses and Prions
- Alphaviruses (Togaviruses), Group A Arboviruses:
- Chikungunya virus (except the vaccine strain 181/25 listed in NIH Guidelines Appendix B-II-D Risk Group 2 (RG2) – Viruses)
- Semliki Forest virus
- St. Louis encephalitis virus
- Venezuelan equine encephalomyelitis virus (except the vaccine strain TC-83, see Appendix B-II-D (RG2))
- Other viruses as listed in BMBL
- Arenaviruses:
- Flexal
- Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)
- Bunyaviruses:
- Hantaviruses, including Hantaan virus
- Rift Valley fever virus
- Coronaviruses, SARS-associated coronavirus (SARS-CoV)
- Flaviviruses (togaviruses), group B arboviruses:
- Japanese encephalitis virus (except those strains listed in NIH Guidelines Appendix B-II-D Risk Group 2 (RG2) – Viruses)
- West Nile virus (WNV)
- Yellow fever virus
- Other viruses as listed in BMBL
- Middle East Respiratory Syndrome coronavirus (MERS-CoV)
- Orthomyxoviruses: Influenza viruses 1918–1919 H1N1 (1918 H1N1), human H2N2 (1957–1968), and highly pathogenic avian influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage (HPAI H5N1)
- Poxviruses: Monkeypox virus
- Prions: Transmissible spongioform encephalopathy (TME) agents (Creutzfeldt-Jacob disease and kuru agents) (see BMBL, for containment instruction)
- Retroviruses:
- Human immunodeficiency virus (HIV) types 1 and 2
- Human T cell lymphotropic virus (HTLV) types 1 and 2
- Simian immunodeficiency virus (SIV)
- Rhabdoviruses: Vesicular stomatitis virus (except those strains listed in NIH Guidelines Appendix B-II-D Risk Group 2 (RG2) – Viruses)
B.2.4 Risk Group 4 Agents
- RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.
- Risk Group 4 Bacterial Agents
- None
- Risk Group 4 Fungal Agents
- None
- Risk Group 4 Parasitic Agents
- None
- Risk Group 4 Viral Agents
- Arenaviruses:
- Guanarito virus
- Lassa virus
- Junin virus (except the candid #1 vaccine strain listed in Appendix B-II-D Risk Group2 (RG2) – Viruses)
- Machupo virus
- Sabia
- Bunyaviruses (Nairovirus): Crimean-Congo hemorrhagic fever virus
- Filoviruses:
- Ebola virus
- Marburg virus
- Flaviruses (Togaviruses), Group B Arboviruses: Tick-borne encephalitis virus complex including Absetterov, Central European encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis viruses
- Herpesviruses (alpha): Herpesvirus simiae (Herpes B or Monkey B virus)
- Paramyxoviruses: Equine morbillivirus (Hendra virus)
- Hemorrhagic fever agents and viruses as yet undefined
- B.2.5 Animal Viral Etiologic Agents in Common Use
- The following list of animal etiologic agents is appended to the list of human etiologic agents. None of these agents are associated with disease in healthy adult humans; however, they are commonly used in laboratory experimental work. A containment level appropriate for RG1 human agents is recommended for their use. For agents that are infectious to human cells, e.g., amphotropic and xenotropic strains of murine leukemia virus, a containment level appropriate for RG2 human agents is recommended.
- Baculoviruses
- Herpesviruses:
- Herpesvirus ateles
- Herpesvirus saimiri
- Marek’s disease virus
- Murine cytomegalovirus
Papilloma viruses:
- Bovine papilloma virus
- Shope papilloma virus
Polyoma viruses:
- Polyoma virus
- Simian virus 40 (SV40)
Retroviruses:
- Avian leukosis virus
- Avian sarcoma virus
- Bovine leukemia virus
- Feline leukemia virus
- Feline sarcoma virus
- Gibbon leukemia virus
- Mason-Pfizer monkey virus
- Mouse mammary tumor virus
- Murine leukemia virus
- Murine sarcoma virus
- Rat leukemia virus
- B.2.6 Murine Retroviral Vectors
- Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than 50% of their respective parental viral genome and that have been demonstrated to be free of detectable replication-competent retrovirus can be maintained, handled, and administered under Biosafety Level (BL) 1 containment.
- B.3 Select Agents and Toxins
- Table B-1 provides the list of select agents and toxins on the National Select Agent Registry (NSAR) established by the Department of Health and Human Services (HHS) Centers for Disease Control and Prevention (CDC) and United States Department of Agriculture (USDA). The most recent online list may be found at http://www.selectagents.gov/index.html. Listed select agents and toxins are categorized as follows:
Agents and toxins that cause disease in humans are listed by HHS CDC as:
- HHS select agents and toxins that affect humans
- Overlap select agents and toxins that affect both (or overlap with) humans and animals
Agents and toxins that cause disease in agricultural animals or plants are listed by USDA as:
- Overlap select agents and toxins that affect humans and animals
- USDA select agents and toxins that affect animals
- USDA Plant Protection and Quarantine (PPQ) select agents and toxins that affect plants
Table B-1. National Select Agent Registry of Select Agents and Toxins |
|
HHS and USDA Select Agents and Toxins 7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73 |
|
HHS Select Agents and Toxins
|
Overlap Select Agents and Toxins
USDA Select Agents and Toxins
USDA Plant Protection and Quarantine (PPQ) Select Agents and Toxins
|
Source: NSAR list updated 10/5/2012 |
- Table B-2 provides additional information, permissible toxin amounts, and synonyms for biological toxins listed on the NSAR of select agents and toxins. A permissible toxin amount is the maximum quantity of biological toxin that can be under the control of a principal investigator at any time without regulation under CDC or USDA.
Table B-2. Additional Information for National Select Agent Registry Toxins |
|||
Name |
Synonyms/Types (Strains)/ Key Words |
CAS Numbers |
Description |
Abrin (100 mg) |
Abrina, Abrin B, Abrin C, Abrin D, Abrin reconstituted (A+B mix), Abrin agglutinin, Toxalbumin |
1393-62-0 (Abrin) 53597-23-2 (Abrin A) 53597-24-3 (Abrin C) |
A powerful phytotoxin present in the seeds of Abrus precatorius (common names include precatory bean, rosemary pea, and jequirity). |
Botulinum neurotoxin
|
Botulinum neurotoxin, Types A, B, C, C1, C2, D, E, F, and G (seven serotypes with a few subtypes). Clostridium botulinum toxin, botulinum toxin, botulinus toxin, botulin toxin |
93384-43-1 (Type A)
|
Produced by the soil bacterium Clostridium botulinum under anaerobic conditions. The most potent toxin known but is heat labile and neutralized by specific antibodies. |
Conotoxins
|
Only short, paralytic alpha conotoxins containing the amino acidsequence X1CCX2PACGX3X4X5X6CX7 are HHS select toxins. The specific conotoxins listed below may or may not meet NSAR’s more-specific 10/5/12 definition of conotoxins: Conotoxins GI, GIA, GII, GIV, GIIIA, GIIIB, GIIIC, GIVA, GVIB, GVIC, Im1, MI,MVIIA,MVIIB, MVIIC,MVIIIv, MVIIDSIA, SVIB (plus more). Conus geographus venom, Conus magus venom, Conus straiatus venom |
81133-24-6 (IV) The above CAS numbers may or may not meet NSAR’s more specific 10/5/12 definition of conotoxins.
|
Small peptide venoms produced by cone shells (Conidia) and marine snails (carnivorous gastropod “cone” mollusks). Venoms vary between species. Act on neuronal communications but each (alpha-mu- and omega-conotoxins) target a different aspect of the process. |
Diacetoxy- |
Diacetoxyscirpenol; Anguidin; Auguidine; Insariotoxin; DAS; 4,15-Diacetoxyscirpen-3-OL; Scirpenetriol 4,15-diacetate; 4 beta,15-diacetoxy-3-alpha-hydroxy-12,13-epoxytrichotech-9-ene |
2270-40-8
|
Trichothecene compound toxins (mycotoxins) produced by various fungus Fusarium, which grow on barley, corn, rye, wheat, etc. |
Ricin
|
Ricinotoxin, Ricinus toxin, Ricin A, Ricin B, Ricin C, Ricin D, Ricin Toxin-Con A, Concanvalin A, Ricin nitrogen, Ricine, Ricin total hydrolysate, Ricinus lectin, Ricnus agglutinin |
9009-86-3 (Ricin), |
A powerful phytotoxin present in the seeds of the castor bean oil plant (Ricinus communis). |
Saxitoxin
|
Mytilotoxin; Saxitoxin hydrate; Saxitoxin hydrochloride; Saxitoxin dihydrochloride; STX dihydrochloride; neo-Saxitoxin (neo-STX); Saxitoxin p-bromo benzenesulfonate; Mytilus californianus poison/toxin; Saxidomas giganteus poison/ toxin; Gonyaulas catenella poison/ toxin; Saxitonin diacetate salt |
35523-89-8 35554-08-6
|
Toxin produced by marine dinoflagellates and cyanobacteria and accumulate in organisms such as mussels (Mytilus), and clams (Saxidomas). |
Staphylococcal enterotoxin
|
Staphylococcus enterotoxins subtypes A,B,C, D, E are HHS select toxins. Enterotoxin F is the Toxic Shock Syndrome “Toxin-1” and is not a HHS select toxin. |
11100-45-1 (Enterotoxin B)
|
Toxin produced by a strain of Staphylococcus aureus. Acts on receptors in gut. |
Tetrodotoxin
|
Fugu poison; fugtoxin; Anhydroepiterodotoxin; Deoxytetrodotoxin; 4-Deoxytetrodotoxin; Deoxyterttoxin; Diateylanhydrotetrdotoxin; Diacetate 4, 9-anhydrotetrodotoxin; Ethoxytetrodotoxin; Maculotoxin; Ethyl tetrodotoxin; 4-Deoxy tetrodotoxin; Spheroidine; Tarichatoxin; 4-amino-4-deoxy, 4,9-Anhydrotetrodotoxin; 8,8-Diacetate 4,9-anhydrotetrodotoxin; tetrodotoxin citrate; TTX; (4-alpha)-4-amino-4-deoxy-tetrodotoxin |
4368-28-9 (tetrodotoxin) 13072-89-4 (4,9-anhydrotetrodotoxin)
|
Highly lethal neurotoxin present in numerous species of puffer fish (Tetraodontoidea) and newts (Tarika). |
T-2 Toxin
|
Toxin T-2; T-2 mycotoxin; T-2 hemisuccinate; T-2 tetraol; T-2 Toxin d3; T-2 Triol; 2,4,5-T-2 ethylhexyl ester; 2,4,5-T-2 methylpropyl ester; Insariotoxin; 12,13-tricothecene; Fusariotoxine T-2; Scirpenol |
21259-20-1 (T-2 Toxin) 34114-99-3 (T-2 tetraol) 120467-83-6 (T-2 Toxind3)
|
Trichothecene compound toxins (mycotoxins) produced by various species of fungus Fusarium, which grows on barley, corn, rye, and wheat. |
- Source: LBNL EHS Division (July 2003), revised to reflect changes to NSAR effective 10/5/2012
- B.4 Plant Pathogens
- This appendix of the Biosafety Manual provides lists of bacterial, fungal, and viral plant pathogens that may be used to identify agents that might be considered plant pathogens. Current USDA Web sites and the USDA permit process may also be used to determine whether the USDA considers agents in specific locations (e.g., California) to be plant pathogens.
- B.4.1 Plant Pathogen Bacteria (by Scientific Name)
- Agrobacterium radiobacter, Agrobacterium rubi, Agrobacterium tumefaciens, Agrobacterium vitis, Burkholderia andropogonis, Burkholderia caryophylli, Burkholderia cepacia, Burkholderia cichorii, Burkholderia corrugata, Burkholderia gladioli pv. gladioli, Clavibacter michiganensis subsp. insidiosus, Clavibacter michiganensis subsp. michiganensis, Clavibacter michiganensis subsp. sepedonicus, Curtobacterium flaccumfaciens pv. flaccumfaciens, Erwinia amylovora, Erwinia carotovora subsp. atroseptica, Erwinia carotovora subsp. carotovora, Erwinia chrysanthemi, Erwinia chrysanthemi pv. chrysanthemi, Erwinia chrysanthemi pv. dieffenbachiae, Erwinia chrysanthemi pv. zeae, Erwinia tracheiphila, Pantoea stewartii subsp. stewartii, Pseudomonas syringae pv. apii, Pseudomonas syringae pv. atrofaciens, Pseudomonas syringae pv. coronafaciens, Pseudomonas syringae pv. glycinea, Pseudomonas syringae pv. lachrymans, Pseudomonas syringae pv. mori, Pseudomonas syringae pv. papulans, Pseudomonas syringae pv. phaseolicola, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. tabaci, Pseudomonas syringae pv. tomato1, Ralstonia solanacearum2, Rhodococcus fascians, Spiroplasma citri, Streptomyces scabies, Xanthomonas campestris pv. armoraciae, Xanthomonas campestris pv. campestris, Xanthomonas campestris pv. carotae, Xanthomonas campestris pv. cucurbitae, Xanthomonas campestris pv. hederae, Xanthomonas campestris pv. juglandis, Xanthomonas campestris pv. papavericola, Xanthomonas campestris pv. pelargonii, Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. raphani, Xanthomonas campestris pv. vitians, Xanthomonas campestris pv. zinniae, Xanthomonas fragariae, Xanthomonas phaseoli pv. alfalfae, Xanthomonas phaseoli pv. begoniae, Xanthomonas phaseoli pv. glycines, Xanthomonas phaseoli pv. phaseoli, Xanthomonas translucens pv. translucens, Xanthomonas vesicatoria.
- B.4.2 Plant Pathogen Fungi (by Scientific Name)
- CHYTRIDIOMYCETES
- Physoderma maydis
- OOMYCETES
- Albugo candida, Peronospora sojae, Peronospora trifoliorum, Peronospora viticola, Phytophthora cactorum, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora citricola, Phytophthora fragariae, Phytophthora infestans, Phytophthora megasperma, Phytophthora megasperma f.sp. medicaginis, Phytophthora rubi s.sp. fragariae, Phytophthora sojae, Plasmodiophora brassicae, Pythium aphanidermatum, Pythium arrhenomanes, Pythium graminicola, Pythium irregulare, Pythium ultimum, Sclerophthora macrospora.
- ASCOMYCETES
- Apiosporina morbosa (black knot), Botryosphaeria obtusa, Botryosphaeria ribis (B. dothidea, B. berengeriana), Claviceps purpurea, Cymadothea trifolii (sooty blotch), Diaporthe phaseolorum, Gaeumannomyces graminis, Gibberella zeae, Glomerella cingulata, Leptosphaerulina trifolii, Monilinia fructicola (Sclerotinia fructicola), Nectria cinnabarina, Ophiostoma ulmi (Ceratocystis ulmi), Pseudopeziza medicaginis, Pseudopeziza trifolii, Sclerotinia sclerotiorum (Whetzelinia sclerotiorum), Sclerotinia trifoliorum, Valsa ambiens, Venturia inaequalis (apple scab), Xylaria polymorpha.
- Powdery Mildews
- Erysiphe graminis, Microsphaera vaccinii (on Ericaceae), Podosphaera clandestina (on Rosaceae), Sphaerotheca Asteraceae, Cucurbitaceae, Scrophulariaceae), Sphaerotheca macularis (on hops and strawberry), Unicinula viticola.
- Coelomycetes
- Colletotrichum acutatum, Colletotrichum coccodes, Colletotrichum destructivum, Colletotrichum fragariae, Colletotrichum gloeosporioides, Colletotrichum graminicola, Colletotrichum trifolii, Macrophomina phaseolina (Macrophoma phaseolina, M. phaseoli, Botryodiplodia phaseoli), Phoma medicaginis, Phomopsis juniperovora, Phomopsis sojae, Phomopsis viticola, Septoria rubi, Septoria tritici, Sphaeropsis sapinea (Diplodia pinea), Stagonospora nodorum (Septoria nodorum), Stenocarpelia maydis (Diplodia zeae, D. zeae-maydis).
- Hyphomycetes
- Alternaria alternata, Alternaria solani, Bipolaris maydis (Heminthosporium maydis, Drechslera maydis), Bipolaris sorokiniana (Helminthosporium sorokiniana, Drechslera sorokiniana), Bipolaris victoriae (Helminthosporium victoriae, Drechslera victoriae), Botrytis cinerea.
- Cercospora medicaginis, Cercospora zeae-maydis, Cladosporium herbarum, Drechslera avenae (on oats, other grasses), Drechslera graminea (on barley, other grasses), Drechslera poae (on grasses), Drechslera teres (on barley, other grasses), Drechslera tritici-repentis (on cereals, other grasses), Exserohilum turcicum (Helminthosporium turcicum, Bipolaris turcicum), Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium equiseti, Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum, Fusarium oxysporum, Fusarium roseum, Fusarium solani, Penicillium expansum, Rhynchosporium secalis, Thielaviopsis basicola, Verticillium albo-atrum, Verticillium dahliae.
- HEMIASCOMYCETES
- Taphrina caerulescens (leaf blister on oak, Ostrya, Rhus), Taphrina communis (plum pocket on Prunus), Taphrina deformans (peach leaf curl).
- BASIDIOMYCETES
- Wood Rotters and Root-Collar Rotters
- Armillaria mellea, Ceratobasidium cerealea, Daedaleopsis confragosa (Daedalea confragosa), Ganoderma applanatum (Fomes applanatus), Ganoderma lucidum, Hirschioporus pargamenus (Trichaptum biformis, Polyporus pargamenus), Laetiporus sulphureus (Polyporus sulphureus), Phellinus gilius, Phellinus robiniae, Schizophyllum commune, Stereum ostrea, Trametes versicolor (Polyporus versicolor, Coriolus versicolor).
- Rusts
- Gymnosporangium clavipes (cedar-quince rust), Gymnosporangium globosum (cedar-hawthorn rust), Gymnosporangium juniperi-virginianae (cedar-apple rust), Puccinia coronata (on Rhamnaceae, Eleganaceae/Poaceae), Puccinia graminis (on Berberis/Poaceae), Puccinia recondita (on Ranunculaceae/Poaceae), Pucciniastrum americanum (late leaf rust on raspberry).
- Smuts
- Tilletia caries (Tilletia tritici), Tilletia laevis (Tilletia foetida), Ustilago avenae, Ustilago hordei, Ustilago tritici, Ustilago zeae.
- Other Basidiomycetes
- Rhizoctonia solani (Thanatephorus cucumeris), Sclerotium rolfsii.
- B.4.3 Plant Pathogen Viruses (Regulated by the State of California)
- Alfalfa mosaic, barley yellow dwarf, bean common mosaic, bean yellow mosaic, beet curly top, beet mosaic, cactus virus X, camellia yellow mottle, carnation mottle, cauliflower mosaic, chrysanthemum mosaic, chrysanthemum virus B, cucumber mosaic, cymbidium mosaic, dasheen mosaic, fig mosaic, impatiens necrotic spot, lettuce big vein, lettuce mosaic, lily symptomless, maize dwarf mosaic, odontoglossum ringspot, papaya ringspot, pepper mottle, plum line pattern, potato leafroll, potato virus S, potato virus X, potato virus Y, prune dwarf, prunus necrotic ringspot, squash mosaic, sugarcane mosaic, tobacco etch, tomato mosaic, tomato spotted wilt, turnip mosaic, watermelon mosaic virus 2, zucchini yellow mosaic.
- B.5 Aerosol Transmissable Pathogens – Laboratory
- A list of Aerosol Transimissble Pathogens- Laboratory can be found in the Cal/OSHA Aerosol Transmissible Diseases Standard Appendix D.
- ____________________
- Appendix C. Laboratory Biosafety Level 1 and Biosafety Level 2 Criteria
- C.1 Introduction and Scope
- This appendix describes criteria for laboratory Biosafety Level 1 (BL1) and BL2 in the same manner and level of detail as presented in Biosafety in Microbiological and Biomedical Laboratories (BMBL), fifth edition. Requirements from the NIH Guidelines and Cal/OSHA Bloodborne Pathogens Standard were also added by LBNL to each BMBL criteria statement as needed to integrate requirements from all of these standards. LBNL requirements were also succinctly added when needed to clarify important requirements or implementation policy specifically related to BMBL criteria statements.
- See Work Process C, Biosafety Principles and Levels, of this manual for additional information on biosafety principles and levels, and Work Process C.4.a, Laboratory Containment Levels, for additional information on laboratory biosafety levels. See Section 5 of this manual for additional information and requirements on controls described in specific criteria statements.
C.2 Laboratory Biosafety Level 1
- Biosafety Level 1 (BL1) is suitable for work involving well-characterized agents that are not known to consistently cause disease in immunocompetent adult humans, and that present minimal potential hazard to laboratory personnel and the environment. BL1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open benchtops using standard microbiological practices. Special containment equipment or facility design is not required but may be used as determined by appropriate risk assessment. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science.
- The following standard practices, safety equipment, and facility requirements apply to BL1:
C.2.1 BL1 Standard Microbiological Practices
The laboratory supervisor and work lead must enforce LBNL institutional policies that control access to the site and laboratory facilities as described in the LBNL Site Security Plan. Policies and practices include, for example, the hosting of visitors and the issuance of gate passes, badges, and/or keys to control access to the site, building, and/or room based on each individual’s business need and experiments in progress. In addition, laboratory areas should have doors for access control.
- Persons must wash their hands: (a) after working with potentially hazardous materials, recombinant materials, and animals; (b) after removing gloves; and (c) before leaving the laboratory.
- Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption are not permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
- Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
- Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware, must be developed and implemented. Whenever practical, laboratory supervisors should adopt improved engineering and work-practice controls that reduce the risk of sharps injuries.
- Precautions must always be taken with sharp items, including those listed below:
- Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
- Used disposable sharps must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal.
- Nondisposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.
- Broken glassware must not be handled directly. Instead, it must be removed by using a brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware whenever possible.
- Perform all procedures to minimize splashes and/or aerosols.
- Decontaminate work surfaces with appropriate disinfectant after completion of work and after any spill or splash of potentially infectious or viable recombinant material.
- Use an effective method to decontaminate all cultures, stocks, and other potentially infectious or recombinant materials before disposal. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
- Materials to be decontaminated outside of the immediate laboratory must be placed in a durable leak-proof container and secured for transport.
- Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
- An effective integrated pest management program is required.
- The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection or ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of childbearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals who have these conditions should be encouraged to identify themselves to the institution’s health care provider for appropriate counseling and guidance.
- C.2.2 BL1 Special Practices
- None required.
- C.2.3 BL1 Safety Equipment (Primary Barriers and Personal Protective Equipment)
- Special containment devices or equipment, such as biosafety cabinets (BSCs), are not generally required.
- Protective laboratory clothing (e.g., coats, gowns, or uniforms) should be worn to prevent contamination of personal clothing.
- Eye protection must be worn in the laboratory and when conducting procedures that have the potential to create splashes of biological materials or other hazardous materials.
- Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Wash hands prior to leaving the laboratory. In addition, BL1 workers should:
- Change gloves when contaminated, when their integrity has been compromised, or when otherwise necessary.
- Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
- Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
- C.2.4 BL1 Laboratory Facilities (Secondary Barriers)
- Laboratories should have doors for access control.
- Laboratories must have a sink for hand washing.
- The laboratory should be designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate.
- Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
- Benchtops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
- Chairs used in laboratory work must be covered with a nonporous material that can be easily cleaned and decontaminated with an appropriate disinfectant.
- Laboratory windows that open to the exterior should be fitted with screens.
- C.3 Laboratory Biosafety Level 2
- Biosafety Level 2 builds upon BL1. BL2 is suitable for work involving agents that pose moderate hazards to personnel and the environment. It differs from BL1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are supervised by scientists competent in handling infectious agents and associated procedures; (2) access to the laboratory is restricted when work is being conducted; and (3) all procedures in which infectious aerosols or splashes may be created are conducted in BSCs or other physical containment equipment.
- The following standard and special practices, safety equipment, and facility requirements apply to BL2:
- C.3.1 BL2 Standard Microbiological Practices
- The laboratory supervisor and work lead must enforce the institutional policies that control access to the site and laboratory facilities as described in the LBNL Site Security Plan. Policies and practices include, for example, the hosting of visitors and the issuance of gate passes, badges, and/or keys to control access to the site, building, and/or room based on each individual’s business need and experiments in progress. Access to the laboratory should be controlled when the laboratory is unoccupied during nonbusiness hours, (e.g., by locking doors to the laboratory areas and/or doors to the building entrance).
- Persons must wash their hands (a) after working with potentially hazardous materials, recombinant materials, and animals; (b) after removing gloves; and (c) before leaving the laboratory.
- Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human consumption are not permitted in laboratory areas. Food must be stored outside the laboratory area in cabinets or refrigerators designated and used for this purpose.
- Mouth pipetting is prohibited. Mechanical pipetting devices must be used.
- Policies for the safe handling of sharps, such as needles, scalpels, pipettes, and broken glassware, must be developed and implemented. Whenever practical, the laboratory supervisor and work lead should adopt improved engineering and work practice controls that reduce risk of sharps injuries. Use of sharps with Risk Group (RG) 2 materials should be restricted and included in the Biological Use Authorization (BUA) as part of the risk assessment.
- Precautions must always be taken with sharp items, including those listed below:
- Careful management of needles and other sharps are of primary importance. Needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal.
- Used disposable sharps must be carefully placed in conveniently located, properly labeled, leak-proof, puncture-resistant, and closable containers used for sharps disposal. Contaminated disposable sharps are disposed of immediately after use in containers that are not overfilled. These containers are closed immediately when full.
- Nondisposable sharps must be placed in a properly labeled, leak-proof, puncture-resistant, hard-walled container for transport to a processing area for decontamination, preferably by autoclaving. In addition, these sharps must not be stored or processed in a manner that requires workers to reach by hand into the containers where these sharps have been placed.
- Broken glassware must not be handled directly. Instead, it must be removed by using a brush and dustpan, tongs, or forceps. Plasticware should be substituted for glassware whenever possible.
- Perform all procedures to minimize the creation of splashes and/or aerosols.
- Decontaminate work surfaces with an appropriate disinfectant after completion of work and after any spill or splash of potentially infectious or viable recombinant material.
- Decontaminate all cultures, stocks, and other potentially infectious or recombinant materials before disposal, using an effective method. Depending on where the decontamination will be performed, the following methods should be used prior to transport:
- Materials to be decontaminated outside of the immediate laboratory must be placed in a durable, leak-proof container and secured for transport.
- Materials to be removed from the facility for decontamination must be packed in accordance with applicable local, state, and federal regulations.
- When infectious agents (i.e., human pathogens) are present or there are organisms that require special provisions for entry (e.g., vaccination), additional biological hazard warning signage is required at the laboratory entrance. This signage must include a biohazard label and the laboratory’s biosafety level; the identity of the agent(s) or the words “Infectious Agent(s)”; the name and telephone number of the supervisor, work lead, principal investigator, or other responsible personnel; and any special requirements or procedures for entering and exiting the laboratory. The Chemical Hygiene and Safety Plan (CHSP) Caution Placard will be used to accomplish these additional signage requirements. Any requirements for posting identities of agents or posting special entry and exit procedures will be specified in the BUA.
- An effective integrated pest management program is required.
- The laboratory supervisor must ensure that laboratory personnel receive appropriate training regarding their duties, the necessary precautions to prevent exposures, and exposure evaluation procedures. Personnel must receive annual updates or additional training when procedural or policy changes occur. Personal health status may impact an individual’s susceptibility to infection, or ability to receive immunizations or prophylactic interventions. Therefore, all laboratory personnel and particularly women of childbearing age should be provided with information regarding immune competence and conditions that may predispose them to infection. Individuals who have these conditions should be encouraged to identify themselves to the institution’s health care provider for appropriate counseling and guidance.
- C.3.2 BL2 Special Practices
- All persons entering the laboratory must be advised of the potential hazards and meet any specific entry/exit requirements as communicated through laboratory door postings or other means. Minimum biosafety hazard advisories include a required biohazard label posted at the entrance to the BL2 laboratory. Any additional biosafety requirements necessary for advising and protecting personnel entering and exiting the area will be specified in the BUA based on a risk assessment.
- Laboratory personnel must be provided with medical surveillance and offered appropriate immunizations for agents handled or potentially present in the laboratory.
- When appropriate, a baseline serum sample should be stored.
- A laboratory-specific biosafety manual must be prepared and adopted as policy, and must be available and accessible.
- The laboratory supervisor must ensure that laboratory personnel demonstrate proficiency in standard and special microbiological practices before working with BL2 agents.
- Potentially infectious materials must be placed in a durable, leak-proof container during collection, handling, processing, storage, or transport within a facility.
- Laboratory equipment should be decontaminated on a routine basis and after spills, splashes, or other potential contamination.
- Spills involving infectious materials must be contained, decontaminated, and cleaned by staff properly trained and equipped to work with infectious material.
- Equipment must be decontaminated before repair, maintenance, or removal from the laboratory.\
- Incidents that may result in exposure to infectious materials must be immediately evaluated and treated according to procedures described in the laboratory biosafety safety manual. All such incidents must be reported to the laboratory supervisor. Medical evaluation, surveillance, and treatment should be provided. Appropriate records should be maintained.
- Animals and plants not associated with the work being performed must not be permitted in the laboratory.
- All procedures involving the manipulation of infectious materials that may generate an aerosol should be conducted within a BSC or other physical containment device.
- C.3.3 BL2 Safety Equipment (Primary Barriers and Personal Protective Equipment)
- Properly maintained BSCs (preferably Class II), other appropriate personal protective equipment, or other physical containment devices must be used whenever:
- Procedures with a potential for creating infectious aerosols or splashes are conducted. These may include pipetting, centrifuging, grinding, blending, shaking, mixing, sonicating, opening containers of infectious materials, inoculating animals intranasally, and harvesting infected tissues from animals or eggs.
- High concentrations or large volumes of infectious agents or organisms containing recombinant DNA are used. Such materials may be centrifuged in the open laboratory using sealed rotor heads or centrifuge safety cups. In this case, the rotor heads and centrifuge cups must be opened inside a BSC.
- Protective laboratory clothing (e.g., coats, gowns, smocks, or uniforms) designated for laboratory use should be worn to prevent contamination of personal clothing and must be worn when working at BL2 or when working with RG2 or other hazardous materials. Remove protective clothing before leaving for nonlaboratory areas (e.g., cafeteria, library, administrative offices). Dispose of protective clothing appropriately, or deposit it for laundering services provided by an LBNL subcontractor. Laboratory clothing must not be taken home.
- Eye protection must be worn in the laboratory. Eye and face protection (goggles, mask, face shield, or other splatter guard) must be used when it is anticipated that splashes, sprays, splatters, or droplets of infectious or other hazardous materials may be generated and could contaminate the eyes, nose, or mouth (e.g., when RG2 microorganisms must be handled outside the BSC or containment device). This eye and face protection must be disposed of with other contaminated laboratory waste or decontaminated before reuse.
- Gloves must be worn to protect hands from exposure to hazardous materials. Glove selection should be based on an appropriate risk assessment. Alternatives to latex gloves should be available. Gloves that were used in BL1 and BL2 work must not be worn outside the laboratory. In addition, BL2 laboratory workers should:
- Change gloves when contaminated, when their integrity has been compromised, or when otherwise necessary. Wear two pairs of gloves when appropriate.
- Remove gloves and wash hands when work with hazardous materials has been completed and before leaving the laboratory.
- Do not wash or reuse disposable gloves. Dispose of used gloves with other contaminated laboratory waste. Hand washing protocols must be rigorously followed.
- Eye, face, and respiratory protection should be used in rooms containing infected animals, as determined by the risk assessment.
- C.3.4 BL2 Laboratory Facilities (Secondary Barriers)
- Laboratory areas should have doors for access and ventilation control, and the doors should be self-closing and have locks designed in accordance with LBNL standards.
- Laboratories must have a sink for hand washing. The sink may be manually, hands-free, or automatically operated. The sink should be located near the exit door.
- The laboratory should be designed so that it can be easily cleaned and decontaminated. Carpets and rugs in laboratories are not permitted.
- Laboratory furniture must be capable of supporting anticipated loads and uses. Spaces between benches, cabinets, and equipment should be accessible for cleaning.
- Benchtops must be impervious to water and resistant to heat, organic solvents, acids, alkalis, and other chemicals.
- Chairs used in laboratory work must be covered with a nonporous material that can be easily cleaned and decontaminated with appropriate disinfectant.
- Laboratory windows that open to the exterior are not recommended. However, if a laboratory does have windows that open to the exterior, they must be fitted with screens.
- BSCs must be installed so that fluctuations of the room air supply and exhaust do not interfere with proper operations. BSCs should be located away from doors, windows that can be opened, heavily traveled laboratory areas, and other possible airflow disruptions.
- Vacuum lines should be protected with high-efficiency particulate air (HEPA) filters or their equivalent. Filters must be replaced as needed. Liquid disinfectant traps may be required.
- An eyewash station must be readily available.
- There are no specific requirements on ventilation systems. However, planning of new facilities should consider mechanical ventilation systems that provide an inward flow of air without recirculation to spaces outside of the laboratory.
- HEPA-filtered exhaust air from a Class II BSC can be safely recirculated back into the laboratory environment if the cabinet is tested and certified at least annually, and operated according to manufacturer’s recommendations. BSCs can also be connected to the laboratory exhaust system by either a thimble (canopy) connection or a direct (hard) connection. Provisions to ensure proper safety cabinet performance and air system operation must be verified.
- A method for decontaminating all laboratory wastes should be available in the facility (e.g., autoclave, chemical disinfection, incineration, or other validated decontamination method).
- ____________________
- Appendix D. Good Microbiological Practice
- D.1 Introduction and Scope
- Good and standard microbiological practices. Source: Berkeley Lab EHS.
- This appendix describes (1) customary principles of good microbiological practice (GMP) and (2) explains the differences between GMP and laboratory biosafety practices defined by the Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) and provided in Appendix C, Laboratory Biosafety Level 1 and Biosafety Level 2 Criteria, of this manual. These GMP principles are guidelines that may be used to control the biosafety and research quality aspects of laboratory work. These guidelines are not biosafety requirements unless other sections of this manual describe them as biosafety requirements.
- The first and most important element of control for research product protection and laboratory containment is strict adherence to (1) GMP and (2) standard microbiological practices and special practices. These sets of practices have different main objectives, but include many overlapping practices and secondary objectives. Both sets of practices should be used when conducting work.
- Good Microbiological Practice (GMP) consists of aseptic techniques and other good microbiological practices that are not uniformly defined but are necessary to prevent contamination of the laboratory with the agents being handled and contamination of the work with agents from the environment. GMP is used to keep the agents being handled inside their primary containers without any other organisms getting in and contaminating the research materials. The main objective of GMP is to ensure that contamination does not affect the research results.
- Standard microbiological practices and special practices are defined by the CDC and NIH, discussed in Work Process C.1, Laboratory Practices, of this program, and listed in detail in Appendix C of this manual. Standard microbiological practices and special practices are used much like GMP to keep agents inside their primary containers. However, the main objective of these practices is to provide safety controls needed to protect workers and the environment from contamination in the event that the agents are accidentally released from their primary container.
- D.2 Good Microbiological Practice
- GMP involves the use of aseptic techniques and other good microbiological practices. These practices and techniques prevent:
- Handled organisms from contaminating the laboratory and
- Organisms in a laboratory environment from contaminating the work.
- Both objectives are important, but the first objective is primarily important for the safety of the worker, while the second objective is mostly important for the quality of the research.
- The principles of GMP should generally be applied to all types of work involving microorganisms and tissue cultures, regardless of containment level.
- D.2.1 Aseptic Technique
- An aseptic technique is a procedure used to grow a microorganism or culture of interest in a clean micro-environment isolated from the outside world. This micro-environment is usually some sort of culture or holding container such as a flask, bottle, or petri dish. The organisms or cells can either be on a solid agar medium or be suspended in a broth, diluent, or other fluid medium.
- Examples of aseptic techniques include ensuring all components of the system are sterile prior to use (e.g., container interior, growth medium, and any items used in manipulation) and using special care and techniques to avoid cross-contamination during the inoculation, incubation, and processing steps. They also include:
- Keeping the container closed except for the minimum time required to introduce or remove materials.
- Holding open containers at an angle whenever possible to prevent contaminants from entering the container.
- Protecting sterile containers from contamination, and working with these containers inside a biosafety cabinet (BSC). When working outside a BSC, a Bunsen burner may be used to flame the opening of the container whenever tops are removed (i.e., passing the opening quickly through the Bunsen flame). The upwards current of hot air created by the Bunsen burner prevents contaminated air or particles from dropping into the culture container when the lid is open.
- Using manipulation techniques that minimize the possibility of cross-contamination (e.g., opening lids with the little finger so that tops are not put down on the work surface).
- Ensuring that all tools (e.g., pipette tips or loops) or other items that may come in contact with the culture are sterile and not contaminated by casual contact with the bench, fingers, or outside of the bottle. Also ensuring that these tools are disposed of or decontaminated immediately after use.
- In addition to aseptic techniques, GMP includes a wide range of other working methods that minimize the cross-contamination of the work and workplace. Examples of these methods are provided in the remaining sections of this appendix.
- D.2.2 Personal Hygiene and Dress
- Wash hands prior to and following manipulations of organisms or cultures and whenever contamination is suspected.
- Wear personal protective equipment (PPE) to protect the worker and to prevent research materials from contamination. Change gloves when contaminated. Routinely clean lab coats or throw away disposable coats.
- Tie back or confine loose or long hair.
- Do not touch the skin, face, or unclean or nonsterile surfaces.
- Keep fingernail tips at a length of one-quarter inch or shorter.
- D.2.3 Area Cleanliness and Organization
- Keep the laboratory and work area clean and organized, such as in the following examples:
- Keep only items necessary for the task in progress on the bench or in the BSC. This practice avoids unnecessary clutter that may collect contaminants, prevent surface disinfection and spill cleanup, and increase the possibility of things getting knocked over.
- Plan and lay out work so that everything needed for a procedure is ready to be handled in a logical order. This practice should allow the worker to sit at the BSC or bench and handle the items efficiently using aseptic techniques.
- Use appropriate chemical antimicrobials (e.g., disinfectants) and decontamination procedures. See Appendix F of this program.
- Wash hands and disinfect work surfaces before and after work.
- Immediately clean spills, and then disinfect the work surface and wash hands.
- Organize the work area when work is complete.
- Avoid putting items on the floor. This practice allows the cleanliness of the floor to be viewed and all parts of the floor to be cleaned routinely, eases spill cleanup, and prevents tripping hazards.
- Routinely clean water baths to minimize microbial contamination of the water.
- Routinely clean laboratory surfaces such as open shelving, benchtops, windowsills, and items on them to prevent accumulation of dust and debris. Store infrequently used items in cabinets and drawers.
- Routinely clean floors and difficult-to-access areas to prevent buildup of dust and debris.
- Routinely clean sink faucets and basins.
- Identify areas and systems in the laboratory and support areas (e.g., wash and autoclave area) for storage and staging of dirty, contaminated, clean, and sterilized items that are being stored, used, or processed for eventual reuse. Ensure everyone understands and follows the system.
- Periodically review items stored in refrigerators and freezers and on shelves and benches. Dispose of items that are no longer needed.
- D.2.4 Biosafety Cabinets and Airborne Contamination
- Use a BSC when needed to protect biological research materials and when procedures may generate biohazardous aerosols. See Appendix E.3, Biosafety Cabinet Work Practices and Procedures, for additional GMP and containment work practices related to work in a BSC.
- Minimize personnel traffic and unnecessary movements around the work area or BSC. Such movements cause area air turbulence that may transport contaminants into the work area and onto the biological materials that need protection. Such movements also disturb clean laminar airflows inside BSCs responsible for containing aerosols and protecting biological materials.
- D.2.5 Manipulation Techniques for Minimizing Aerosols
- Manipulation techniques should be used to minimize the possibility of producing aerosols. Examples include:
- Mixing by gentle rolling and swirling rather than vigorous shaking (to avoid frothing)
- Pipetting by putting the tip into a liquid or onto a surface prior to gently ejecting the pipette contents (to avoid bubbling and splashing)
- Placing containers in very close proximity to each other when transferring liquids between them (to avoid drops that fall and splash)
- Allowing loops to cool down after incineration or flaming before using the loop (to avoid sizzling)
- Not overfilling centrifuge tubes (to avoid leakage into centrifuge)
- Slowly removing tube caps or stoppers
- Not popping caps off of tubes
- Carrying and storing cultures (e.g., bottles and plates) in racks and spill-proof containers (to prevent dropping and breakage)
- D.2.6 Worker Qualifications
- Workers who handle microorganisms and cultures should have sufficient technical competence, training, and experience in GMP and containment practices. In addition, workers should use GMP and biosafety containment in anticipation of unexpected hazards when handling microorganisms and cultures (including Risk Group 1). Workers should conservatively approach their safety by assuming, for example, that an unexpected pathogen may exist or contaminate the culture; a pathogen may be unintentionally cultured; the disease potential of the agent may be altered under laboratory conditions; or exposure to an RG1 agent may cause an opportunistic infection.
- D.2.7 Microbial Contamination Checks
- Routine microbial contamination checks should be incorporated into protocols and undertaken at various stages of experiments. Examples of contamination checks include:
- Taking a loopful of fluid from the vessel and plating (or streaking) it out onto a nonselective solid nutrient medium to look for single colonies.
- Incubating culture samples at a suitable temperature (usually 30°C) to allow growth of contaminants originating from the general environment and human sources.
- After incubation, examining plates for evidence of any contamination as indicated by colony types.
- The purity of a liquid culture can also be obtained by microscopic examination. This is done by placing a loopful of the culture on a microscope slide. The slide is then examined wet either by phase contrast microscopy, or by fixed or Gram staining. Contaminant organisms should be instantly and clearly visible.
- Contamination checks are particularly useful in evaluating GMP competence. Workers with poor aseptic techniques will have frequent contamination problems, while workers skilled in GMP will have problems only occasionally. It is important to recognize that poor practices not only result in contaminated cultures, but may also result in spreading biological materials and contamination to work surfaces and workers in the laboratory.
- D.3 References
- University of Edinburgh, Health and Safety, “Good Microbiological Practice and Containment,” Web page information from the Health and Safety Department, August 2003.
- ____________________
- Appendix E Biosafety Cabinets
- E.1 Introduction and Scope
- Biosafety cabinet. Source: Berkeley Lab EHS.
- Biological safety cabinets or biosafety cabinets (BSCs) are hoods with high-efficiency particulate air (HEPA) filters that are designed to provide personnel, environmental, and product protection when appropriate practices and procedures are followed. Key BSC information and requirements are summarized in Work Process D.6.d.ii, Hoods and Biosafety Cabinets, of this program. This appendix provides the following information and requirements on BSCs:
- Classifications
- Work practices and procedures
- Decontamination
- Installation and engineering
- Testing and certification
- Information in this appendix primarily contains excerpts from Appendix A – Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety Cabinets of Biosafety in Microbiological and Biomedical Laboratories (BMBL), fifth edition. This appendix also reiterates LBNL policies presented in Work Process D.6.d.ii, Hoods and Biosafety Cabinets, of this program.
- E.2 Biosafety Cabinet Classifications
- Three primary types of BSCs have been developed to meet varying research and clinical needs. These primary BSC types are designated as Class I, II, and III. Class II BSCs are also further subdivided into different Class II types. Tables E-1 and E-2 summarize the similarities and differences in the types of protection and physical characteristics of different classes of BSCs. The sections following these tables summarize and illustrate the characteristics of BSC classes used at LBNL. This information should be used in BSC selection and risk assessment.
- Table E-1. Protection Offered by Classes of Biosafety Cabinets
Biological |
Protection Provided |
|
||
Personnel |
Product |
Environmental |
BSC Class |
|
Biosafety Level (BL) 1 to 3 |
Yes |
No |
Yes |
I |
BL1 to 3 |
Yes |
Yes |
Yes |
II (A1, A2, B1, B2) |
BL4
|
Yes |
Yes |
Yes |
III II – when used in room with suit |
* Source: adapted from BMBL, fifth edition, Appendix A, Table 1. |
- Table E-2. Characteristics of Biosafety Cabinet Classes
BSC Class, Type
|
Face Velocity |
Airflow Pattern |
Applications |
|
Nonvolatile Toxic Chemicals and |
Volatile Toxic Chemicals and Radionuclides
|
|||
I
|
75 |
In at front through HEPA to the outside or into the room through HEPA (Figure 1) |
Yes |
When exhausted outdoors 1,2 |
II, A1 |
75
|
70% recirculated to the cabinet work area through HEPA. 30% balance can be exhausted through HEPA back into the room or to outside through a canopy unit. |
Yes (minute amounts) |
No |
II, A2 |
100
|
Similar to II, A1, but has 100 linear fpm intake air velocity and plenums are under negative pressure to room (Figure 2). Exhaust air can be ducted to outside through a canopy unit (Figure 3). |
Yes
|
When exhausted outdoors (formerly “B3”) (minute amounts) 1,2 |
II, B1 |
100
|
30% recirculated, 70% exhausted. Exhaust cabinet air must pass through a dedicated duct to the outside through a HEPA filter (Figure 4). |
Yes
|
Yes (minute amounts)1,2
|
II, B2 |
100
|
No recirculation. Total exhaust to the outside through a HEPA filter. |
Yes |
Yes (small amounts) 1,2 |
III |
N/A
|
Supply air is HEPA filtered. Exhaust air passes through two HEPA filters in series and is exhausted to the outside via a hard connection (Figure 5). |
Yes |
Yes (small amounts) 1,2
|
Footnotes: |
* Source: adapted from BMBL, fifth edition, Appendix A, Table 2. |
- E.2.1 Class I Biosafety Cabinet
- The Class I BSC provides personnel and environmental protection, but no product protection. It is similar in air movement to a chemical fume hood, but has a HEPA filter in the exhaust system to protect the environment. Figure 1 shows a diagram of a Class I BSC.
Figure 1. Class I BSC. (A) front opening, (B) sash, (C) exhaust HEPA filter, and (D) exhaust plenum. Note: The cabinet needs to be hard connected to the building exhaust system if toxic vapors are to be used. Source: BMBL, fifth edition, Appendix A. |
- E.2.2 Class II Biosafety Cabinet
- Class II BSCs (Types A1, A2, B1, and B2) provide personnel, environmental, and product protection. Airflow is drawn into the front grille of the cabinet, providing personnel protection. In addition, the downward laminar flow of HEPA-filtered air provides product protection by minimizing the chance of cross-contamination across the work surface of the cabinet. Because cabinet exhaust air is passed through a certified HEPA filter, the exhaust air is particulate-free (environmental protection), and may be recirculated to the laboratory (i.e., Type A1 and A2 BSCs only) or discharged from the building via the exhaust duct system and a canopy connection. Exhaust air from Type B1 and B2 BSCs must be discharged to the outdoors via a hard duct connection. Figure 2 shows a diagram of a Class II, Type A2 BSC, which is the most common type of BSC at LBNL. Figure 3 shows a diagram of a canopy (or thimble) unit that is normally required when connecting a Class II, Type A1 or A2 BSC to an exhaust duct system. Figure 4 shows a Class II, Type B2 BSC, which is also used at LBNL. Installation of a Class II, Type B2 BSC typically requires a hard duct connection to the exhaust system without a canopy or thimble unit connection.
- HEPA filters are effective at trapping particulates and thus infectious agents but do not capture volatile chemicals or gases. Only Type A2 exhausted or Types B1and B2 BSCs exhausting to the outside should be used when working with volatile toxic chemicals, but amounts must be limited. See Table 2 for additional information.
Figure 2. Class II, Type A2 BSC. Tabletop model. (A) front opening, (B) sash, (C) exhaust HEPA filter, (D) supply HEPA filter, (E) positive-pressure common plenum, (F) negative-pressure plenum. Unless it is connected to the building exhaust system, the Class II, Type A2 BSC is not equivalent to what was formerly called a Class II, Type B3 BSC. Note: The Class II, Type A2 BSC should be canopy-connected to the exhaust system. Diagram source (left): adapted from BMBL, fifth edition, Appendix A. Picture source (right): Berkeley Lab EHS. |
Figure 3. Canopy (Thimble) Unit. Canopy (thimble) units for connecting a Class II, Type A1 or A2 BSC to the exhaust duct system. (A) balancing damper, (B) flexible connector to exhaust system, (C) cabinet exhaust HEPA filter housing, (D) canopy unit, (E) BSC. Note: There is a one-inch gap between (D) the canopy unit and (E) the exhaust filter housing through which room air is exhausted. Source: adapted from BMBL, fifth edition, Appendix A. |
Figure 4. Class II, Type B2 BSC. (A) Front opening, (B) sash, (C) exhaust HEPA filter, (D) supply HEPA filter, (E) negative-pressure exhaust plenum. Note: The cabinet exhaust needs to be hard connected to the building exhaust system. Diagram source (left): BMBL, fifth edition, Appendix A. Picture source (right): Berkeley Lab EHS. |
- E.2.3 Class III Biosafety Cabinet
- A standard Class III BSC (Figure 5) is designed for working with highly infectious microbiological agents and conducting hazardous operations. It is a gas-tight enclosure with an un-openable view window that provides maximum protection for the environment and the worker. Access for passage of materials into the cabinet is through a chemical dunk tank accessible through the cabinet floor, or a double-door pass-through box (e.g., an autoclave) that can be decontaminated between uses. Reversing that process allows materials to be safely removed from the Class III BSC. Both supply and exhaust air pass through a HEPA filter on a Class III cabinet. Exhaust air must pass through two HEPA filters, or a HEPA filter and an air incinerator, before discharge to the outdoors. Airflow is maintained by a dedicated, independent exhaust system exterior to the cabinet, which keeps the cabinet under negative pressure (minimum pressure of 0.5 inches of water gauge). Some Class III BSCs may not have all of these controls, based on the risk assessment conducted (e.g., types of materials and manner of work).
- Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet. The gloves allow users to directly manipulate materials isolated inside and prevent the user’s direct contact with hazardous materials. Depending on the design of the cabinet, the supply HEPA filter provides particulate-free airflow within the work environment. Laminar airflow is not a characteristic of a Class III cabinet.
Figure 5. Class III BSC. (A) Glove ports with O-ring for attaching arm-length gloves to cabinet, (B) sash, (C) exhaust HEPA filter, (D) supply HEPA filter, (E) double-ended autoclave or pass-through box. The cabinet exhaust needs to be hard connected to an independent dedicated exhaust system. The exhaust air must be double HEPA filtered or HEPA filtered and incinerated. Source: adapted from BMBL, fifth edition, Appendix A. |
- E.2.4 Clean Benches (Not BSCs)
- Horizontal and vertical laminar flow clean benches are shown in Figures 6 and 7. These units may provide protection for the product, but are not considered safety hoods or BSCs and must not be used for infectious or toxic materials or when a hood or BSC is needed to protect the worker.
Figure 6. Horizontal Laminar Flow Clean Bench. (A) Front opening, (B) grille, (C) supply HEPA filter, (D) plenum, (E) blower, (F) grille. Source: BMBL, fifth edition, Appendix A. |
Figure 7. Vertical Laminar Flow Clean Bench. (A) Front opening, (B) sash, (C) supply HEPA filter, (D) blower.
|
- E.3 Biosafety Cabinet Work Practices and Procedures
- This section discusses in detail standard work practices and procedures for investigators working in a Class II BSC. In general, these practices and procedures are important for protection of the worker or the product, but the importance of each practice or procedure for the safety of the worker often depends on the nature of biological materials and the work being conducted. A shorter list of key BSC work practices and procedures is provided in Appendix D.
- Back to Top
- E.3.1 Preparing for BSC Work
- This section discusses preparing for work within a Class II BSC.
- Air Current Disruptions. Preparing a written checklist of materials necessary for a particular activity and placing necessary materials in the BSC before beginning work minimizes the number and extent of air curtain disruptions compromising the fragile air barrier of the cabinet. The rapid movement of a worker’s arms in a sweeping motion into and out of the cabinet will disrupt the air curtain and compromise the partial containment barrier provided by the BSC. Moving arms slowly in and out and perpendicular to the face while opening the cabinet will reduce this risk. Other personnel activities in the room (e.g., rapid movements near the face of the cabinet, walking traffic, room fans, open/closing room doors, etc.) may also disrupt the cabinet air barrier.
- Personal Protective Equipment (PPE). Eye protection and laboratory coats buttoned over street clothing must be worn. Latex, vinyl, nitrile, or other suitable gloves must be worn to provide hand protection. Higher levels of PPE can be included as determined by an individual risk assessment. For example, a solid front, back-closing laboratory gown provides better protection of personal clothing than a traditional laboratory coat and is a recommended practice at when working in a BSC at BL3.
- Body and Material Positioning. Before beginning work, the BSC user should adjust the stool height so that his/her face is above the front opening. Manipulation of materials should be delayed for approximately one minute after placing the hands/arms inside the cabinet. This allows the cabinet to stabilize, the user to “air sweep” his or her hands and arms, and to allow time for turbulence reduction. When the user’s arms rest flatly across the front grille, the arms may occlude the grille opening, and room air laden with particles may flow directly into the work area rather than being drawn down through the front grille. Raising the arms slightly will alleviate this problem. The front grille must not be blocked by towels, research notes, discarded plastic wrappers, pipetting devices, etc. All operations should be performed on the work surface at least four inches from the front grille. If there is a drain valve under the work surface, it should be closed prior to beginning work in the BSC.
- Materials or equipment placed inside the cabinet may cause disruption of the airflow, resulting in turbulence, possible cross-contamination and/or breach of containment. Extra supplies (e.g., additional gloves, culture plates or flasks, culture media) should be stored outside the cabinet. Only the materials and equipment required for immediate work should be placed in the BSC.
- Purge and Decontamination. If the cabinet has been shut down, the blowers should be operated at least four minutes before beginning work to allow the cabinet to “purge.” This purge will remove any suspended particulates in the cabinet. The work surface, the interior walls (except the supply filter diffuser), and the interior surface of the window should be wiped with 70% ethanol (EtOH), a 1:100 dilution of household bleach (i.e., 0.05% sodium hypochlorite), or other disinfectant as determined by the investigator to meet the requirements of the particular activity. When bleach is used, a second wiping with sterile water is needed to remove the residual chlorine, which may eventually corrode stainless-steel surfaces. Wiping with nonsterile water may recontaminate cabinet surfaces, a critical issue when sterility is essential (e.g., maintenance of cell cultures).
Prepare the BSC for Work. Source: Berkeley Lab EHS |
- Similarly, the surfaces of all materials and containers placed into the cabinet should be wiped with 70% EtOH to reduce the introduction of contaminants to the cabinet environment. This simple step will reduce the introduction of mold spores and thereby minimize the contamination of cultures. The further reduction of microbial load on materials to be placed or used in BSCs may be achieved by periodic decontamination of incubators and refrigerators.
- Back to Top
- E.3.2 Material Placement inside the BSC
- Material placement and work in a BSC. Source: Berkeley Lab EHS.
- This section covers placement of materials inside the BSC.
- Surface Towels. Plastic-backed absorbent towels can be placed on the work surface but not on the front or rear grille openings. The use of towels facilitates routine cleanup and reduces splatter and aerosol generation during an overt spill. The used towel can be folded and placed in a biohazard bag or other appropriate receptacle when work is completed.
- Inside Materials and Sash. All materials should be placed as far back in the cabinet as practical, toward the rear edge of the work surface and away from the front grille of the cabinet (Figure 8). Similarly, aerosol-generating equipment (e.g., vortex mixers, tabletop centrifuges) should be placed toward the rear of the cabinet. This placement of materials and equipment allows the BSC’s downward flow of laminar air to flow with minimal turbulence and to be captured by the front and rear grilles . Bulky items such as biohazard bags, discard pipette trays, and vacuum collection flasks should be placed to one side of the interior of the cabinet. If placing those items in the cabinet requires opening the sash, make sure that the sash is returned to its original position before work is initiated. The correct sash position (usually 8 or 10 inches above the base of the opening) should be indicated on the front of the cabinet. On most BSCs, an audible alarm will sound if the sash is in the wrong position while the fan is operating.
- Practices That Do Not Interfere with BSC Operation. Certain common practices could interfere with the operation of the BSC. For example, the biohazard collection bag should not be taped to the outside of the cabinet. Additionally, upright pipette collection containers should not be used in BSCs nor placed on the floor outside the cabinet, as the frequent inward/outward movement needed to place objects in these containers is disruptive to the integrity of the cabinet air barrier and can compromise both personnel and product protection. Only horizontal pipette discard trays containing an appropriate chemical disinfectant should be used within the cabinet. Furthermore, potentially contaminated materials should not be brought out of the cabinet until they have been surface-decontaminated. Alternatively, contaminated materials can be placed into a closable container for transfer to an incubator, autoclave, or another part of the laboratory.
- Back to Top
- E.3.3 Operations within a Class II BSC
- Splatters and Aerosols. Many procedures conducted in BSCs may create splatters or aerosols. Good microbiological techniques should always be used when working in a BSC. For example, techniques used to reduce splatter and aerosol generation will also minimize the potential for personnel exposure to infectious materials manipulated within the cabinet. Class II cabinets are designed so that horizontally nebulized spores introduced into the cabinet will be captured by the downward flowing cabinet air within 14 inches of travel. Therefore, as a general rule of thumb, keeping clean materials at least one foot away from aerosol-generating activities will minimize the potential for cross-contamination.
- Work Flow:
- The work flow should be from “clean to dirty” (see Figure 8). Materials and supplies should be placed in the cabinet in such a way as to limit the movement of “dirty” items over “clean” ones. Several measures can be taken to reduce the chance for cross-contamination of materials when working in a BSC. Opened tubes or bottles should not be held in a vertical position. Investigators working with petri dishes and tissue culture plates should hold the lid above the open sterile surface to minimize direct impaction of downward air. Bottle or tube caps should not be placed on the towels. Items should be recapped or covered as soon as possible.
Figure 8. Typical Work Layout Inside a BSC. Shown above is a typical layout for working “clean to dirty” within a Class II BSC. Clean cultures (left) can be inoculated (center); contaminated pipettes can be discarded in the shallow pan, and other contaminated materials can be placed in the biohazard bag (right). This arrangement is reversed for left-handed persons. Source: adapted by Berkeley Lab EHS from BMBL, fifth edition, Appendix A. |
- <class=”c2″>Burners and Open Flames. Open flames are not required in the near-microbe-free environment of a BSC. On an open bench, flaming the neck of a culture vessel will create an upward air current, which prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, creates turbulence that disrupts the pattern of HEPA-filtered air being supplied to the work surface and may cause fires. When deemed absolutely necessary, touch-plate microburners equipped with a pilot light to provide an on-demand flame should be used. These burners will minimize internal cabinet air disturbance, heat buildup, and fire risk. The burner must be turned off when work is completed. Small electric “furnaces” are also available for decontaminating bacteriological loops and needles, and are preferable to an open flame inside the BSC. Disposable or recyclable sterile loops should be used whenever possible.
A fire inside a BSC occurred when the gas rubber hose connected to a Touch-O-Matic Bunsen burner melted and gas in the hose ignited. Brookhaven National Laboratory, Lessons Learned 2002-CHBNL-MED-0003 (July 23, 2007). |
BSC fire. Source: Stanford University, Use of open flames in Cabinets/Tissue Culture Hoods (May 29, 2003). |
- The following are examples of burners and heaters that could be used in a biosafety cabinet if other sterile techniques are not feasible:
- Burner: Touch-O-Matic Bunsen Burner
Simply depress ON/OFF platform with side of hand to release gas stream, and continuous pinpoint pilot light ignites gas to produce full flame. Release platform and flame goes out to conserve gas. To produce continuous flame, depress platform, then turn it slightly; reverse process to turn off flame. - Heater: Bacti-Cinerator
Infrared heat chamber sterilizes loops, needles, and culture tubes in five to seven seconds. Suitable for anaerobic procedures in chambers and hoods. Electric heat source eliminates hazards from gas and open flames. Within six minutes of activation, the interior of the ceramic cone reaches an optimum sterilizing temperature of 815°C (1,500°F). A prominent light indicates when the unit is in operation. Weighted cast aluminum stand includes handy spaces for storage of six inoculating loop handles. Electrical: 120V, 50/60Hz. UL listed. Unit is not intended for use with scalpels, forceps, or sharp instruments.
Touch-O-Matic Bunsen Burner. Source: Fisher Scientific (May 2010). |
Bacti-Cinerator. Source: VWR (May 2010). |
- Aspirator Bottles or Suction Flasks. Aspirator bottles or suction flasks should be connected to an overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or equivalent filter (see Figure 9). This combination will provide protection to the central building vacuum system or vacuum pump, as well as to the personnel who service this equipment. Inactivation of aspirated materials can be accomplished by placing sufficient chemical decontamination solution into the flask to inactivate the microorganisms as they are collected. Once inactivation occurs, liquid materials can be disposed of as noninfectious waste.
- Aspirator bottles that collect Risk Group (RG) 1 or RG2 biological materials that do not contain RG2 infectious agents may be placed outside the BSC as long as the aspirator bottles are placed inside a secondary spill tray.
Figure 9. Aspiration and House Vacuum System Protection. Shown below is one method to protect a house vacuum system during aspiration of infectious fluids. The left suction flask (A) is used to collect the contaminated fluids into a suitable decontamination solution; the right flask (B) serves as a fluid overflow collection vessel. An in-line HEPA filter (C) is used to protect the vacuum system (D) from aerosolized microorganisms. A spill tray (E) should be used when the flasks are outside the BSC. Source: adapted by Berkeley Lab EHS from BMBL, fifth edition, Appendix A. |
- E.4 Biosafety Cabinet Decontamination and Moves
- E.4.1 Cabinet Surface Decontamination
- Cabinet Surfaces. With the cabinet blower running, all containers and equipment should be surface-decontaminated and removed from the cabinet when work is completed. At the end of the workday, the final surface decontamination of the cabinet should include a wipe-down of the work surface, the cabinet’s sides and back, and the interior of the glass. If necessary, the cabinet should also be monitored for radioactivity and decontaminated when necessary. Investigators should remove their gloves and gowns in a manner that prevents the contamination of unprotected skin and aerosol generation, and wash their hands as the final step in safe microbiological practices. The cabinet blower may be turned off or left on after these operations are completed.
- Small Spills. Small spills within the operating BSC can be handled immediately by removing the contaminated absorbent paper towel and placing it into the biohazard bag or receptacle. Any splatter onto items within the cabinet, as well as the cabinet interior, should be immediately cleaned up with a towel dampened with an appropriate decontaminating solution. Gloves should be changed after the work surface is decontaminated and before placing clean absorbent towel in the cabinet. Hands should be washed whenever gloves are changed or removed.
- Large Spills. Spills large enough to result in liquids flowing through the front or rear grilles require more extensive decontamination. All items within the cabinet should be surface decontaminated and removed. After ensuring that the drain valve is closed, decontaminating solution can be poured onto the work surface and through the grille(s) into the drain pan.
- Decontamination Time and Cleanup. Twenty to 30 minutes is generally considered an appropriate contact time for decontamination, but this varies with the disinfectant and the microbiological agent. Manufacturer’s directions should be followed. The spilled fluid and disinfectant solution on the work surface should be absorbed with paper towels and discarded into a biohazard bag. The drain pan should be emptied into a collection vessel containing disinfectant. A hose barb and flexible tube should be attached to the drain valve and be of sufficient length to allow the open end to be submerged in the disinfectant within the collection vessel. This procedure serves to minimize aerosol generation. The drain pan should be flushed with water and the drain tube removed.
- Radioactive Materials. Should the spilled liquid contain radioactive material, a similar procedure can be followed. Radiation safety personnel should be contacted for specific instructions.
- Work Surface, Grille, and Drain Pan Cleaning. Periodic removal of the cabinet work surface and/or grilles after the completion of drain pan decontamination may be justified because of dirty drain pan surfaces and grilles, which ultimately could occlude the drain valve or block airflow. However, extreme caution should be observed while wiping these surfaces to avoid injury from broken glass and sharp metal edges. Always use disposable paper towels and avoid applying harsh force. Wipe dirty surfaces gently. Never leave paper towels on the drain pan because the paper could block the drain valve or the air passages in the cabinet.
- E.4.2 Internal Cabinet Gaseous Decontamination
- BSCs that have been used for work involving infectious materials must be decontaminated before HEPA filters are changed or internal repair work is done. Before a BSC is relocated, a risk assessment considering the agents manipulated within the BSC must be performed to determine the need and method for decontamination. LBNL policy requires that BSCs and their filters be decontaminated with a gaseous decontaminant prior to being moved or internal repair work is conducted, unless approved by the Biosafety Officer. The most common decontamination method uses formaldehyde gas, although more recently, hydrogen peroxide vapor and chlorine dioxide gas have been used successfully.
- E.5 Biosafety Cabinet Installation and Engineering
- Room Ventilation and Secondary Barriers. Whereas BSCs are considered to be the primary safety barrier for manipulation of infectious materials, the laboratory room itself is considered to be the secondary safety barrier. Inward directional airflow is established by exhausting a greater volume of air than is supplied to a given laboratory and by drawing makeup air from the adjacent space. This directional air flow into the room should generally be accomplished at BL2 (see Work Process D.6.d.1, Room Ventilation, of this program). The air balance for the entire facility should be established and maintained to ensure that air flows from areas of least to greatest potential contamination.
- The room exhaust system should be sized to handle both the room and all containment devices vented through the system. Adequate supply air must be provided to ensure appropriate function of the exhaust system. The facility engineer must be consulted before locating a new cabinet requiring connection to the building exhaust system. Right angle bends, long horizontal runs, and transitional connections within the systems will add to the demand on the exhaust fan. The building exhaust air should be discharged away from supply air intakes to prevent re-entrainment of laboratory exhaust air into the building air supply system.
- Utility Services. Utility services needed within a BSC must be planned carefully. Protection of vacuum systems must be addressed (Figure 9). Electrical outlets inside the cabinet must be protected by ground fault circuit interrupters and should be supplied by an independent circuit. When propane or natural gas is provided, a clearly marked emergency gas shutoff valve must be installed outside the cabinet for fire safety. All nonelectrical utility services should have exposed, accessible shutoff valves. The use of compressed air within a BSC must be carefully considered and controlled to prevent aerosol production and reduce the potential for vessel pressurization.
- BSC UV light. Source: Berkeley Lab EHS.
- Ultraviolet (UV) Lamps. UV lamps are not required in BSCs nor are they necessary. If installed, UV lamps must be cleaned weekly to remove any dust and dirt that may block the germicidal effectiveness of the ultraviolet light. The lamps should be checked weekly with a UV meter to ensure that the appropriate intensity of UV light is being emitted. UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure, which can burn the cornea and cause skin cancer. If the cabinet has a sliding sash, close the sash when operating the UV lamp.
- BSC Placement. BSCs were developed as workstations to provide personnel, environmental, and product protection during the manipulation of infectious microorganisms. Certain considerations must be met to ensure maximum effectiveness of these primary barriers. Whenever possible, adequate clearance should be provided behind and on each side of the cabinet to allow easy access for maintenance and to ensure that the cabinet air recirculated to the laboratory is not hindered. A 12- to 14-inch clearance above the cabinet may be required to provide for accurate air velocity measurement across the exhaust filter surface and for exhaust filter changes. When the BSC is hard ducted or connected by a canopy unit to the ventilation system, adequate space must be provided so that the configuration of the duct work will not interfere with airflow. The canopy unit must provide adequate access to the exhaust HEPA filter for testing.
- The ideal location for the biological safety cabinet is away from the entry (i.e., the rear of the laboratory away from traffic), since people walking parallel to the face of a BSC can disrupt the air curtain. The air curtain created at the front of the cabinet is quite fragile, amounting to a nominal inward and downward velocity of 1 mph. Open windows, air supply registers, portable fans, or laboratory equipment that create air movement (e.g., centrifuges, vacuum pumps) should not be located near the BSC. Similarly, chemical fume hoods must not be located close to BSCs.
- E.6 Biosafety Cabinet Testing and Certification
- Class II BSCs are the primary containment devices that protect the worker, product, and environment from exposure to microbiological agents. BSCs used for BL1, BL2, or other safety levels must be tested and certified before initial use, after being moved, and on a nominal one-year cycle. This testing must verify that BSC operation is in accordance with the National Sanitation Foundation (NSF) and American National Standard Institute (ANSI) 49 standard (e.g., NSF/ANSI 49-2012 Biosafety Cabinetry: Design, Construction, Performance, and Field Certification) and be performed by experienced and qualified personnel. This testing ensures the balance of inflow and exhaust air, distribution of air onto the work surface, integrity of the cabinet and the filters, and other BSC features. The LBNL Environment/Health/Safety (EHS) Industrial Hygiene Group manages surveys and tests of BSCs through the LBNL ventilation safety program and qualified vendors contracted to test BSCs (see Work Process D.6.d.ii of this program).
- Biosafety cabinet survey and certification labels. Source: Berkeley Lab EHS.
- New BSCs or other laminar flow clean benches that need to contain airborne hazards should be constructed, tested, and certified by the manufacturer to meet the NSF/ANSI 49 standard. Specific manufacturers and models that are certified to this standard are listed by NSF as NSF Certified Biosafety Cabinetry.
- E.7 References
- Biosafety in Microbiological and Biomedical Laboratories (BMBL), fifth edition. Appendix A, Primary Containment for Biohazards: Selection, Installation, and Use of Biological Safety Cabinets
- National Sanitation Foundation and American National Standard Institute standard, NSF/ANSI 49-2012 Biosafety Cabinetry: Design, Construction, Performance, and Field Certification
- Brookhaven National Laboratory, Lessons Learned 2002-CHBNL-MED-0003, Propane Fire within a Biological Safety Cabinet, July 23, 2007
- Stanford University, Use of open flames in Cabinets/Tissue Culture Hoods, May 29, 2003
- ____________________
- Appendix F. Decontamination and Antimicrobials
- F.1 Introduction and Scope
- This appendix primarily provides information and guidance on decontamination principles, decontamination terms, and the variety of chemical and physical agents used to decontaminate. In a few cases, requirements are stated using the words should or must. See Section 5.7 of this manual for requirements and additional information regarding decontamination, waste, and decommissioning. Information used to develop this appendix was taken from a wide variety of Web pages and documents. Primary sources are listed in the reference section at the end of this appendix.
- F.2 Decontamination Principles and Terms
- Decontamination is a process that uses an antimicrobial to reduce or inactivate biological contaminants or components to an acceptable level so as to reduce or eliminate the possibility of transmitting pathogens to undesired hosts. An antimicrobial is the chemical or physical agent that is used in a decontamination process to prevent microbial growth. Prevention of microbial growth and pathogen transmission is needed to control contamination of the work and prevent disease in hosts such as laboratory workers, the general public, and other organisms in the environment. The decontamination process, level, antimicrobial, frequency, and specific method are based on the work activity, agents that need inactivation, and decontamination objective or requirements.
- Sterilization, disinfection, sanitization, and antisepsis are decontamination processes that result in different levels of decontamination or decontamination of different types of objects. These processes are discussed in Section F.2.1 below. A variety of terms are also used to describe the antimicrobials that are used in sterilization, disinfection, sanitization, and antisepsis. These antimicrobial terms are discussed in Section F.2.2 below.
- F.2.1 Decontamination Processes and Levels
- Sterilization processes: autoclaving or disposal as medical/biohazardous waste. Source: Berkeley Lab EHS.
- F.2.1.1 Sterilization
- Sterilization is the process of completely destroying all living microorganisms and viruses on an object. Any item, device, or solution is considered to be sterile when it is completely free of all living microorganisms and viruses. Sterility is an absolute term (an item is either sterile or it is not), but sterilization procedures must be defined to achieve sterility. A sterilization procedure is a treatment process to which an item is subjected after which the probability of a microorganism or virus (including a high number of bacterial endospores) surviving on the item is less than one in one million. This level of killing efficacy is referred to as the sterility assurance level.
- Sterilization can be accomplished by heat (e.g., autoclave or incineration), ethylene oxide gas, hydrogen peroxide gas, plasma, ozone, and radiation. Solid biohazardous waste is typically sterilized prior to disposal.
- F.2.1.2 Disinfection
- Disinfection is generally a less lethal process than sterilization. Disinfection is the process of generally eliminating nearly all recognized pathogenic microorganisms but not necessarily all microbial forms (e.g., bacterial spores) on inanimate objects (e.g., work surfaces, equipment). Disinfection does not ensure “overkill” and therefore lacks the margin of safety achieved by sterilization procedures. Longer disinfection times or higher concentrations of disinfectant may be needed if the effectiveness of a disinfection procedure is reduced significantly by a number of factors such as:
- More resistant microorganisms (especially bacterial spores)
- Higher microbial concentrations
- Presence of more organic matter (e.g., soil, feces, or blood)
- Rougher surfaces or more porous equipment or material
- Lower temperatures
Surface disinfectants and disinfection processes. Lower-level disinfection of a laboratory bench top with 70% ethanol. Intermediate-level disinfection of a biosafety cabinet with fresh 1% household bleach and animal cage shelves with chlorine dioxide solution. Source: Berkeley Lab EHS. |
- Disinfection may involve chemical or physical agents, but the term disinfection more commonly implies the use of chemical germicides or disinfectants on inanimate objects. See Section F.2.2 below for additional explanation of germicides and disinfectants.
- Disinfection is a process that reduces the level of microbial contamination, but there is a broad range of activity that extends from sterility at one extreme to a minimal reduction in the number of microbial contaminants at the other. By definition, chemical disinfection and in particular, high-level disinfection differs from chemical sterilization by its lack of sporicidal power. This is an oversimplification of the actual situation because a few chemical germicides used as disinfectants do, in fact, kill large numbers of spores even though high concentrations and several hours of exposure may be required. Nonsporicidal disinfectants may differ in their capacity to accomplish disinfection or decontamination. Some germicides rapidly kill only the ordinary vegetative forms of bacteria such as staphylococci and streptococci, some forms of fungi, and lipid-containing viruses, whereas others are effective against such relatively resistant organisms as Mycobacterium tuberculosis var. bovis, nonlipid viruses, and most forms of fungi.
- Levels of chemical disinfection and activity levels for chemical disinfectants (or germicides) on inanimate surfaces may be used to assist in categorizing and selecting disinfection methods and disinfectants. Levels of chemical disinfection are categorized in Table F-1, and activity levels of selected disinfectants are shown in Table F-2.
- Table F-1. Levels of Chemical Disinfection
Level |
Level Definition and Description |
High |
High-level disinfection kills vegetative microorganisms and inactivates viruses, but not necessarily high numbers of bacterial spores. Such disinfectants are capable of sterilization when the contact time is relatively long (e.g., six to 10 hours). As high-level disinfectants, they are used for relatively short periods of time (e.g., 10 to 30 minutes). These chemical germicides are potent sporicides and in the United States are classified by the Food and Drug Administration (FDA) as sterilants/disinfectants. They are formulated for use on medical devices, but not on environmental surfaces such as laboratory benches or floors. |
Intermediate |
Intermediate-level disinfection kills vegetative microorganisms, including Mycobacterium tuberculosis, all fungi, and inactivates most viruses. Chemical germicides used in this procedure often correspond to Environmental Protection Agency (EPA)-approved hospital disinfectants that are also tuberculocidal. They are used commonly in laboratories for disinfection of laboratory benches and as part of detergent germicides used for housekeeping purposes. |
Low |
Low-level disinfection kills most vegetative bacteria except M. tuberculosis and some fungi, and inactivates some viruses. The EPA approves chemical germicides used in this procedure in the U.S. as hospital disinfectants or sanitizers. |
- Source: adapted from Biosafety in Microbiological and Biomedical Laboratories (BMBL), fifth edition, Appendix B.
- Table F-2. Activity Levels of Selected Liquid Germicidesa
Procedure/Product |
Aqueous Concentration |
Disinfection Activity Level |
Sterilization |
||
Glutaraldehyde |
Variable |
N/A |
Hydrogen peroxide |
6–30% |
N/A |
Formaldehyde |
6–8% b |
N/A |
Chlorine dioxide |
Variable |
N/A |
Peracetic acid |
Variable |
N/A |
Disinfection |
||
Glutaraldehyde |
Variable |
High to intermediate |
Ortho-phthalaldehyde |
0.5% |
High |
Hydrogen peroxide |
3 to 6% |
High to intermediate |
Formaldehyde |
1 to 8% |
High to low |
Chlorine dioxide |
Variable |
High |
Peracetic acid |
Variable |
High |
Chlorine compoundsc |
500 to 5,000 mg/L available chlorine (or 1 to 10% household beach in water) |
Intermediate |
Alcohols(ethyl,isopropyl)d |
70% |
Intermediate |
Phenolic compounds |
0.5 to 3% |
Intermediate to low |
Iodophor compoundse |
30 to 50 mg/L free iodine up to 10,000 mg/L available iodine 0.1 to 0.2% |
Intermediate to low |
Quaternary ammonium Compounds |
|
Low |
Source: adapted from BMBL, fifth edition, Appendix B. Footnotes: This list of chemical germicides centers on generic formulations. A large number of commercial products based on these generic components can be considered for use. Users should ensure that commercial formulations are registered with the EPA or by the FDA.
|
- An understanding of the resistance of organisms to chemical germicides should also be considered when selecting the disinfection methods and disinfectants. Table F-3 shows the resistance of selected organisms to decontamination, from most to least resistant.
- Table F-3. Descending Order of Organism Resistance to Germicidal Chemicals
Descending Order of Resistance to Germicides |
Agent Type |
BACTERIAL SPORES Bacillus subtilis, Clostridium sporogenes |
|
MYCOBACTERIA Mycobacterium tuberculosis var. bovis, nontuberculous mycobacteria |
|
NONLIPID OR SMALL VIRUSES Poliovirus, Coxsackievirus, Rhinovirus |
|
FUNGI Trichophyton spp., Cryptococcus spp., Candida spp.
|
|
VEGETATIVE BACTERIA Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella choleraesuis, Enterococci
|
|
LIPID OR MEDIUM-SIZE VIRUSES Herpes simplex virus, cytomegalovirus, respiratory syncytial virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Hantavirus, Ebola virus
|
- Source: adapted from BMBL, fifth edition, Appendix B
- Note: There are exceptions to this list. Pseudomonas spp. are sensitive to high-level disinfectants, but if they grow in water and form biofilms on surfaces, the protected cells can approach the resistance of bacterial spores to the same disinfectant. The same is true for resistance to glutaraldehyde by some nontuberculous mycobacteria, some fungal ascospores of Microascus cinereus and Cheatomium globosum, and the pink-pigmented Methylobacteria. Prions are also resistant to most liquid chemical germicides and are discussed in the last part of this section.
- F.2.1.3 Sanitization
- Restroom sanitization with a disinfectant Source: Berkeley Lab EHS.
- Sanitization is the process of generally reducing microorganisms by the use of general cleaning agents. Sanitization is less effective than disinfection at reducing the number of microorganisms. General cleaning of laundry or laboratory, restroom, room, and equipment surfaces with soap and water or another cleaning agent are examples of sanitization. A particular cleaning method might use a chemical germicide or disinfectant, but the cleaning process is considered sanitization if the process only generally reduces the number of microorganisms. See Section F.2.2 below for additional explanation of germicides and disinfectants.
- In the food industry, the term sanitization has a more specific meaning. According to the California Retail Food Code (CRFC), sanitization means the application of cumulative heat or chemicals on cleaned food-contact surfaces that, when evaluated for efficacy, is sufficient to yield a reduction of five logs, which is equal to a 99.999% reduction, of representative disease microorganisms of public health importance.
Research sanitization processes: Laboratory coat, glassware, and animal cage washing. Fermenter cleaning using a clean-in-place process unit. Source: Berkeley Lab EHS and Public Affairs. |
- F.2.1.4 Antisepsis
- Antiseptic for animal surgery. Source: Berkeley Lab EHS.
- Antisepsis is the application of a liquid antimicrobial chemical to human or animal living tissue. The purpose of antisepsis is to prevent sepsis by destroying potentially infectious organisms or by inhibiting their growth and multiplication. Sepsis is the presence of infectious organisms in the blood or other tissue of the body. No sporicidal activity is implied. Examples of antisepsis include application of a germicide to the injection site on a research animal, and handwashing with germicidal solution. With handwashing, the objective includes preventing the spread of infectious or contaminating agents for safety and quality control.
- F.2.2 Antimicrobial Categories
- Chemical or physical agents or substances that can decontaminate under ideal conditions have specific terms with specific meanings. The broadest term for such agents is the term antimicrobial. Antimicrobial is a chemical or physical agent that can prevent microbial growth either by some static action or by the direct killing of microbes. Categories of antimicrobials include:
- Sterilant. An antimicrobial chemical or physical agent that is capable of killing all microbes, including their spores, to the sterility assurance level.
- Germicide. An antimicrobial substance or physical agent that kills microbes. Germicides are a broader category of antimicrobials than disinfectants, since some germicides are active against endospores and viruses. Germicides, which are also known for the specific microorganisms they kill, end with the suffix –cidal (e.g., bacteriocide, sporicide, fungicide, virucide).
- Disinfectant. A chemical germicide or physical agent that is applied to inanimate objects to kill microbes, but is not capable of killing endospores, some viruses, or mycobacterium. Disinfectants are typically chemical germicides.
- Antiseptic. A disinfecting chemical agent applied to living tissue and used to prevent sepsis. Antiseptics are a subset of disinfecting chemical agents. A few agents are suitable as both disinfectants and antiseptics, although most disinfectants are too harsh for use on delicate skin.
- F.2.3 Antimicrobial Selection and Registered Disinfectants
- Antimicrobial capabilities and conditions of use. Source: Berkeley Lab EHS.
- When using a chemical or physical antimicrobial to ensure decontamination is accomplished for biosafety purposes (i.e., protection of workers, public, agriculture, or environment):
- There should be information indicating that the selected antimicrobial will be effective when used in a certain manner for the biological materials or agents and equipment or surfaces that need to be decontaminated; and
- The antimicrobial should be used in accordance with its antimicrobial activity capabilities and conditions of use.
- Antimicrobial information in this appendix, information provided by manufacturers (e.g., labels or technical specifications), and other information may be used for selecting and using the appropriate antimicrobial. Selecting a commercially available chemical antimicrobial product registered with the EPA or cleared by the FDA and using the product within its manufacturer-specified limits also ensure effective decontamination. The following lists of antimicrobials registered with EPA and FDA are available online:
- Selected EPA-registered Disinfectants including sterilizers, tuberculocides, and antimicrobial products against certain human public health bacteria and viruses
- FDA-Cleared Sterilants and High-Level Disinfectants with General Claims for Processing Reusable Medical and Dental Devices
- Cal/OSHA Bloodborne Pathogens (BBPs) Standard requires that work surfaces contaminated with BBP material (as defined in Work Process B.3.f, Bloodborne Pathogens and Human Materials, of this program) must be cleaned with an “appropriate disinfectant.” Appropriate disinfectants include:
- Household bleach (i.e., approximately 5.25% sodium hypochlorite) diluted to concentrations ranging from 1% (1:100) to 10% (1:10) in water
- EPA-registered products as sterilants (List A)
- EPA-registered products as tuberculocides (List B)
- EPA-registered products effective against HIV/HBV (List D), or
- FDA-cleared sterilants and high-level disinfectants
- Any of the above products are considered effective when used according to the manufacturer’s instructions, provided the surfaces have not become contaminated with agents, or volumes or concentrations of agents for which higher level disinfection is recommended. Also note that the EPA lists contain the primary registrants’ products only. The same formulation is frequently repackaged and renamed and distributed by other companies. These renamed products will not appear on the list, but their EPA Registration Number must appear on the label. Products cleared solely by the FDA will not have an EPA Number.
- F.3 Chemical Antimicrobials
- This section summarizes basic types and characteristics of antimicrobials that are chemical agents. Section F.4 below summarizes antimicrobials that are physical agents.
- All chemical antimicrobials harm microorganisms in some manner, but different chemical antimicrobials have different mechanisms of action. Mechanisms of harm include protein denaturation, membrane disruption, nucleic acid damage, and inhibition of metabolism. Chemical antimicrobials that are summarized in this section include surfactants, halogen-containing compounds, alcohols, phenol and phenol derivatives, oxidizing agents, and alkylating agents.
- F.3.1 Surfactants (Soaps and Detergents)
- A surfactant is a surface active agent that is usually an organic compound that possesses both hydrophilic (water-loving) and lipophilic (fat-liking) properties that make the compound soluble in water and lipids. Surfactants therefore increase the solubility of lipids in water solutions and increase the ability of water solutions to wet (i.e., move across or penetrate) lipid surfaces. Soaps and detergents are examples of surfactants.
- F.3.1.1 Soaps
- Soap is sodium or potassium salts of fatty acids. Soaps are therefore alkaline (pH greater than 7). Soaps either harm bacteria that are sensitive to high pH, or remove pathogens from surfaces by cleaning the surface.
- F.3.1.2 Detergents and Quaternary Ammonium Compounds
- Detergent is a synthetic surfactant. A detergent may be cationic (positively charged) or anionic (negatively charged). Cationic detergents are better at inactivating bacteria than anionic detergents.
- One commonly used type of cationic detergent disinfectant is a quaternary ammonium compound. Quaternary ammonium compound or quat is a cationic detergent compound derived from ammonia by replacing the hydrogen atoms with organic radicals, and the compound is especially important as surface-active agents or disinfectants, or in drugs. Quats have strong surface activity and can be used for general cleaning and low-level disinfection. Quaternary ammonium compounds are also:
- Active against Gram-positive bacteria and lipid-containing viruses. They are less active against Gram-negative bacteria and are not active against nonlipid-containing viruses and bacterial spores.
- Less effective or inactivated by organic materials, soaps or anionic detergents, or salts of metals found in water. Quats are often mixed with another agent to overcome some of these problems.
- Relatively nontoxic (e.g., can be used for general cleaning and food equipment) and have built-in cleaning properties.
- Odorless but act as a deodorizer.
- Effective at temperatures up to 212°F.
- More effective in alkaline than in acid solutions.
- Typically nonirritating to the skin when used in proper dilution, but prolonged skin or eye contact should be avoided.
- Stable in storage.
- F.3.2 Halogens (Chlorine and Iodine)
- Halogens are a group of elements on the periodic table. Chlorine and iodine are two halogens that are routinely used as antimicrobials.
- F.3.2.1 Chlorine and Sodium Hypochlorite
- Chlorine-containing solutions are commonly used disinfectants, and sodium hypochlorite in the form of household bleach is the most common solution used for chlorine disinfection. These solutions have broad-spectrum antimicrobial activity, but their decay rates and corrosive nature limit their use. The following bullets provide additional information:
- Concentrations and Effectiveness. Chlorine-containing solutions have broad spectrum activity, but the concentration of the chlorine-active ingredient in the solution at time of use affects germicidal activity. Low concentrations of available chlorine (2 to 500 ppm) are active against vegetative bacteria, fungi, and most viruses. Effectiveness increases with concentration of available chlorine. Rapid sporicidal action can be obtained at about 2,500 ppm.
- Active Ingredient Decay. The chlorine-active ingredient typically decays or is consumed. Decay or decomposition typically occurs over time and is accelerated by unfavorable storage conditions. Chlorine is also consumed by excess organic materials. Use of sufficient concentrations and quantity of chlorine, along with precleaning items to be disinfected, ensures sufficient chlorine is available for disinfection.
- Corrosiveness. Chlorine-containing solutions are strong oxidizers and are very corrosive to personnel and some surfaces. Personnel handling these solutions must wear required hand, eye, and body protection (see Work Process D.4, Personal Protective Clothing and Equipment, of this manual). Surfaces such as stainless steel may be corroded and should be wiped or rinsed with water following disinfection.
- One of the most common and effective disinfectants used in the laboratory is sodium hypochlorite (NaOCl) in water or “bleach.” Household bleach is a water-based solution of sodium hypochlorite with a typical concentration of 5.25% by weight (or 52,500 ppm) of the active sodium hypochlorite ingredient. Commercial supplies are also available in the 12 to 15% dilution range, but household bleach is typically sufficient for laboratory use. Many brands and formulations of bleach are registered with the EPA as a disinfectant that is effective against bloodborne and other common human pathogens (see Section F.2.3 above). Clorox® is the best-known brand of bleach in the U.S.
- Common applications and mixtures of household bleach are listed below.
- Work Surfaces and Equipment. Hard work surfaces and equipment may be disinfected with 1% solution of fresh household bleach (or 500 ppm sodium hypochlorite). A 1% household bleach solution can be made by mixing 1 part household bleach with 99 parts water, or 1/8 to 1/4 cup household bleach with water in a gallon container, or 10 ml of household bleach with water in a 1 L container. Contact time for bleach is generally considered to be the time it takes the product to air dry.
- Spills and Liquid Waste. Biohazardous spills and liquid waste may be decontaminated by adding household bleach to water or the liquid to be decontaminated until a 10% concentration of household bleach is achieved (or 5,000 ppm sodium hypochlorite). A 10% household bleach solution can be made by mixing one part household bleach with 9 parts water, or 1.5 cups household bleach with water in a gallon container, or 100 ml of household bleach with water in a 1 L container. The bleach should remain in contact with the spill or waste material for approximately 20 minutes to ensure adequate germicidal action. See Appendix G of this manual for additional information on spill cleanup.
- Sodium hypochlorite solutions are not very stable, and the antimicrobial activity of the chlorine typically decays over time. This decay is accelerated by unfavorable storage conditions and must be compensated by using fresh supplies and mixing fresh solutions. Favorable storage conditions include: temperatures between 50 and 70°F, plastic container (not metal or glass), opaque container (to minimize exposure to light), and closed container (to minimize exposure to air). According to Clorox, manufactured bottles of bleach (a) can be stored for about six months under favorable conditions (after this time, bleach will begin to degrade at a rate of 20% each year until it has totally degraded to salt and water) and (b) should be disposed of after three months if full-strength bleach is required. Since bleach antimicrobial activity decays over time, bleach solutions must be sufficiently fresh so that the solution to be used for decontamination has sufficient antimicrobial activity. Fresh solutions of diluted household bleach should be mixed daily for proper disinfection of work surfaces.
How to mix, use, and store bleach so that it is effective
Household bleach is a water-based solution of sodium hypochlorite (NaOCl) with a typical concentration of 5.25% by weight of the NaOCl active ingredient. Manage bleach’s decay in antimicrobial activity by:
|
||||||
Source: Berkeley Lab EHS |
- F.3.2.2 Iodine and Iodophors
- Iodine is another halogen that is routinely used as an antimicrobial (at 70 to 150 ppm total iodine). Iodine has properties similar to chlorine. Iodophor is a preparation containing iodine complexed with a solubilizing agent, such as a surfactant or povidone (a type of water soluble polyvinyl polymer). The resulting iodophor is a water-soluble material that increases penetration (as a surfactant) and slows the release of free iodine over long periods (as a disinfectant) when in solution. Iodophors are prepared by mixing iodine with the solubilizing agent. Wescodyne® is a common laboratory disinfectant iodophor.
- Additional properties of iodophors include:
- Rapid germicidal action. Effective against vegetative bacteria, Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, and tubercle bacilli. Poor activity against bacterial spores.
- Most effective in acid solutions.
- Should not be used in hot water, since iodine is vaporized at 120 to 125°F. For optimal germicidal activity, dilute with warm acidic water. Resulting solutions are less stable but have a higher germicidal activity.
- Effectiveness reduced by organic matter (but not as much as hypochlorites).
- Stable in storage if kept cool and tightly covered.
- Relatively harmless and nontoxic to humans.
- The solution has germicidal activity if the color is brown or yellow.
- Solutions of sodium thiosulfate can be used to inactivate iodophors and remove iodophor stains.
- Iodophors may also be used as antiseptics. Betadine and isodine are examples of antiseptic iodophors. Iodine may also be used in an alcohol solution (i.e., or tincture) as an antiseptic.
- F.3.3 Alcohols
- 70% Ethanol. Source: Berkeley Lab EHS.
- Ethyl or isopropyl (rubbing) alcohol concentrations of 70 to 90% in water are good general-use disinfectants with some limitations. Alcohol-water mixtures are more penetrating than pure alcohols, and therefore provide better disinfection. Alcohol concentrations above 90% are less effective than 70 to 90% concentrations.
- Alcohols have some positive and negative characteristics, including:
- Alcohols are effective against a broad spectrum of bacterial species and many viruses, but they are less active against nonlipid viruses and ineffective against bacterial spores.
- Alcohols evaporate quickly and leave no residue. These characteristics often make alcohols convenient and efficient, but provide limited penetration and disinfection time.
- F.3.4 Phenol and Phenol Derivatives (Phenolics)
- Phenol and phenol derivatives (or phenolics) come in various concentrations ranging mostly from 5 to 10% phenol-based compounds. These disinfectants are especially useful for disinfecting materials contaminated with organic materials and contaminated surfaces. Lysol® is an example of a phenol-based disinfectant.
- Additional properties of phenol and phenol derivatives include the following:
- Effective at killing Gram-negative and Gram-positive bacteria including Mycobacterium tuberculosis, fungi, and lipid-containing viruses. Not active against spores or most nonlipid viruses.
- Low solubility in water unless combined with detergent.
- Stable in storage.
- Less adversely affected by organic matter than other common disinfectants.
- Effective over a relatively large pH range.
- Prolonged contact deteriorates rubber.
- Can cause skin and eye irritation.
- Not for use on food contact surfaces.
- Some phenolics are mild enough for use as antiseptics whereas others are too harsh or otherwise dangerous to be employed on living tissue.
- F.3.5 Oxidizing Agents (Hydrogen Peroxide)
- Hydrogen peroxide is an oxidizing agent and may be used as a liquid or vapor antimicrobial. Hydrogen peroxide vapor may be used for decontamination of equipment such as biosafety cabinets or high-containment (Biosafety Level 3) rooms that may be sealed during the decontamination process.
- F.3.6 Alkylating Agents (Formaldehyde, Glutaraldehyde, Ethylene Oxide)
- Formaldehyde, glutaraldehyde, and ethylene oxides are alkylating agents. These agents add carbon-containing functional groups to biological molecules.
- F.3.6.1 Formaldehyde
- Formaldehyde may be used as a liquid or gaseous antimicrobial. When used as a liquid, formaldehyde may be mixed with water as formalin or mixed with alcohol. Formaldehyde is also a human carcinogen, creates respiratory problems, and has a very low occupational exposure ceiling and short-term exposure limits that are approximately equal to the odor threshold.
- Additional information on formaldehyde antimicrobials are listed below:
- Formalin is 37% solution of formaldehyde in water. Dilution of formalin to 5% results in an effective disinfectant. A concentration of 8% formaldehyde exhibits good activity against vegetative bacteria, spores, and viruses.
- Formaldehyde and alcohol solutions (8% formaldehyde in 70% alcohol) are considered very good disinfectants because of their effectiveness against vegetative bacteria, fungi, spores, and viruses. This is the disinfectant of choice for many applications.
- Formaldehyde gas may be generated by heat-accelerated depolymerization of paraformaldehyde flakes. The resulting gas may be used to decontaminate equipment such as biosafety cabinets that may be sealed prior to decontamination.
- F.3.6.2 Glutaraldehyde
- Gluteraldehyde may be used for cold sterilization of equipment (e.g., medical) that cannot be steam sterilized, but sterilization often requires many hours of exposure. Two percent solutions exhibit good activity against vegetative bacteria, spores, and viruses. Its use, however, must be limited and controlled due to its toxic properties and ability to damage the eyes.
- Glutaraldehyde is slightly acidic in aqueous solution and typically used at ambient temperature. When these solutions are adjusted by sodium bicarbonate (or other buffers) to a pH of 7.5 to 8.5, glutaraldehyde is considered to be activated and the antimicrobial activity enhanced. Activated glutaraldehyde has limited stability after activation.
- F.3.6.3 Ethylene Oxide
- Ethylene oxide is a gaseous chemical antimicrobial used to sterilize laboratory, medical, and pharmaceutical products and equipment that would be damaged by high-temperature steam sterilization (e.g., prepackaged plastic petri dishes). This gas is especially useful because it penetrates very well into small crevices.
- F.4 Physical Antimicrobials
- This section summarizes basic types and characteristics of antimicrobials that are physical agents. Physical antimicrobials summarized in this section include dry heat, wet heat, ultraviolet radiation, ionizing radiation, visible light, and filtration.
- F.4.1 Heat
- Dry heat (e.g., oven) and moist heat (e.g., autoclave) may be used to sterilize materials and equipment. The following principles and comparisons generally apply to sterilization with dry and moist heat:
- Moist heat is more effective than dry heat at a given temperature or length of exposure.
- Moist heat is more penetrating than dry heat.
- Temperature and length of exposure are inversely related, and penetration is critical.
- Temperature and length of exposure needed to achieve sterilization are inversely related (i.e., lower temperatures require longer exposure times).
- Time to achieve sterilization does not start until heat has penetrated into the item and the required temperature in the item has been achieved.
- F.4.1.1 Dry Heat (Baking and Incineration)
- Dry heat sterilization may include baking or incineration:
- Baking in an oven to achieve sterilization typically requires 171°C for at least one hour, 160°C for at least two hours, or 121°C for at least 16 hours.
- Incineration may also be used to achieve dry heat sterilization. Examples include off-site incineration of biohazardous or pathological waste by an LBNL subcontractor or heating an inoculating loop in an infrared heat chamber at 815°C (1,500°F).
- Specific times and temperatures must be determined for each type of material being sterilized. Generous safety factors are usually added to allow for variables that can influence the efficiency of dry heat sterilization, such as:
- The moisture of the sterilization environment as well as the moisture history of organisms prior to heat exposure.
- The heat transfer properties and the spatial configuration or arrangement of articles in the load.
- F.4.1.2 Wet Heat (Boiling and Autoclaving)
- Use of wet heat may include boiling an item in water or processing the item in an autoclave. Boiling water is a common means of applying moist heat, but boiling does not kill endospores and all viruses. Boiling water is 100°C (212°F) at standard atmospheric pressure. Higher wet-heat temperatures and sterilization efficacy may be achieved with a pressurized autoclave.
- Autoclaves are commonly used to sterilize laboratory equipment or materials such as glassware, media, reagents, or waste. See Section F.5 below for general information and guidelines on autoclave principles, operation, and maintenance.
- F.4.2 Ultraviolet (UV) Radiation
- UV radiation or UV light is electromagnetic radiation with a wavelength shorter than that of visible light but longer than X-rays. UV wavelengths are in the range of 10 nanometers (nm) to 400 nm, and their energy ranges from 3 electron volts (eV) to 124 eV. UV radiation is so named because the spectrum consists of electromagnetic waves with frequencies that are higher than visible violet light.
- F.4.2.1 UV Light Health Effects and Categories
- UV radiation may affect or damage the skin and eyes depending on the wavelength, intensity, and duration of exposure. Other organs are typically not affected because UV light does not penetrate deeply into tissue. Acute effects to the skin and eyes are generally not permanent but can be quite painful.
- The UV spectrum is divided into three wavelength bands primarily based on their biological effects:
- UVA (315 to 400 nm) is long-wave UV or “back light” and is used in dentistry and tanning. UVA rays can penetrate the middle layer of skin (dermis) and cause darkening and toughening of the skin. Overexposure to UVA has also been associated with suppression of the immune system and cataract formation.
- UVB (280 to 315 nm) is medium-wave UV and is used for fade testing and photocuring of plastics. UVB rays reach the outer layer of skin (epidermis) and cause skin burns, erythma (reddening of the skin), and darkening of the skin. Prolonged exposures increase the risk of skin cancer.
- UVC (100 to 280 nm) is short-wave UV and is used as a germicidal (e.g., inside biosafety cabinets). UVC poses the most risk to skin. Although UVC from the sun is absorbed by the atmosphere, manmade sources of UVC need to restrict their intensity and control exposure.
- Electromagnetic spectrum. Source: CCOHS, OSH Answers, Physical Agents, Ultraviolet Radiation (February 2010).
- UV light that penetrates skin. Source: FDA, Radiation-emitting Products, Ultraviolet Radiation (February 2010). Blue UV light inside a biosafety cabinet. Source: Berkeley Lab EHS.
- The eyes are particularly sensitive to UV radiation. Even a short exposure of a few seconds can result in painful but temporary inflammatory conditions known as photokeratitis and conjunctivitis. Examples of eye disorders resulting from UV exposure include “flash burn,” “ground-glass eye ball,” “welder’s flash,” and “snow blindness.” The symptoms are pain, discomfort similar to the feeling of sand in the eye, and an aversion to bright light.
- The eyes are most sensitive to UV radiation from 210 nm to 320 nm (UVC and UVB). Maximum absorption by the cornea occurs around 280 nm. UVA absorption by the lens may be a factor in producing a cataract (a clouding of the lens in the eye).
- All wavelengths less than 320 nm (UVB and UVC) are actinic, which means they are capable of causing chemical reactions. Wavelengths below 180 nm are of little practical biological significance since the atmosphere readily absorbs them.
- F.4.2.2 Biosafety Cabinet UV Light
- BSC UV light. Source: Berkeley Lab EHS.
- Long-term exposure to UV light may be used for disinfecting surfaces and air; however, UV light is not recommended or necessary for use inside biosafety cabinets (BSCs). This is because UV light is limited by many factors (see bulleted list below) as a disinfectant and harmful to human tissue. Other means of disinfection (e.g., chemical) are recommended for use inside BSCs.
- UV light’s ability to disinfect inside BSCs is limited by a number of factors including:
- Penetration: UV light lacks penetrating power. Microorganisms beneath dust particles or beneath the work surface are not affected by the UV radiation.
- Relative Humidity: Humidity decreases the effectiveness of UV light. Antimicrobial effects of UV light drops off precipitously above 70% relative humidity.
- Temperature and Air Movement: Optimum temperature for UV light output is 77 to 80°F. Temperatures below this optimum temperature result in reduced output of the antimicrobial wavelength. Moving air tends to cool the lamp below its optimum operating temperature and results in reduced output.
- Lamp Cleanliness: Dust and dirt can block the antimicrobial effectiveness of UV lights. UV lamps need to be cleaned weekly with an alcohol and water mixture.
- Lamp Age: The intensity of UV light emitted from UV lamps decreases with age, and bulb ratings (hours of use) may vary by manufacturer. UV lamps need to be checked periodically (approximately every six months) to ensure the intensity and wavelength of UV light needed for antimicrobial activity is being emitted.
- See Appendix E, Section E.5, of this manual for additional information on using UV light inside BSCs. If UV light is used as an antimicrobial but is not a required biosafety control, then maintenance and testing of the UV lights is not required for biosafety purposes. For example, germicides are used as the primary means of BSC disinfection, so maintenance and testing of the UV light inside the BSC is not required for biosafety purposes.
- F.4.3 Ionizing Radiation
- Ionizing radiation is radiation of sufficiently high energy to cause ionization in the medium through which it passes. This radiation may be of a stream of high-energy particles (e.g. electrons, protons, alpha particles) or short-wavelength electromagnetic radiation (e.g., ultraviolet, X-rays, gamma rays). This type of radiation can cause extensive damage to the molecular structure of a substance either as a result of the direct transfer of energy to its atoms or molecules, or as a result of the secondary electrons released by ionization. The effect of ionizing radiation in biological tissue can be very serious, usually as a consequence of the ejection of an electron from a water molecule and the oxidizing or reducing effects of highly reactive species. Biological effects on living cells may include DNA damage and mutations.
- Ionizing and nonionizing radiation. Source: Wikipedia, “Nonionizing Radiation” (February 2010).
- Different types of ionizing radiation display different degrees of penetration and may be used to sterilize equipment (e.g., medical instruments) or biological materials (e.g., inside human cadaver bones). Use of ionizing radiation as an antimicrobial requires established and specialized methods known to sterilize specific items.
- F.4.4 Visible Light
- Strong visible light can decrease bacterial viability. Drying laundry on a clothesline is an example of disinfection by using detergents and strong visible light.
- F.4.5 Filtration (HEPA Filters)
- Filtration is used as an antimicrobial treatment for air and liquids.
- High-efficiency particulate air (HEPA) filters are used to filter air flowing into aseptic areas (e.g., the work area inside a BSC) and out of potentially contaminated areas (e.g., exhaust from a BSC). See Work Process D.6.d.ii, Hoods and Biosafety Cabinets, and Appendix E of this program for additional HEPA filter and BSC information.
- Filtration is commonly used when materials are heat labile, but sterilization is not necessarily achieved unless the filter has very small filter pores. Smaller filter pores will also slow filtration speed.
- F.5 Autoclave Sterilization and Safety
- Tabletop and floor-standing autoclaves. Source: Berkeley Lab EHS.
- This section provides general information and guidelines on autoclave principles, operation, and maintenance typically needed to sterilize materials or equipment and ensure operator safety. An autoclave is a piece of equipment with a chamber that is used to sterilize items by applying wet heat (i.e., high-pressure steam) at temperatures above the normal boiling point of water and pressures above normal atmospheric pressure.
- Autoclaves are used to sterilize laboratory equipment or materials such as glassware, media, reagents, or waste. Autoclaves are commonly used because they are a dependable means of achieving the necessary level of killing efficacy (or sterility assurance level) for most biological materials. In addition, autoclaves do not generate other chemical antimicrobial waste or sources of contamination. See Section F.2.1.1 for general information on sterilization and killing efficacy.
- Autoclaves must be operated and monitored properly to achieve sterility and safety. Operator safety is a concern because autoclaves may pose physical hazards (e.g., heat, steam, pressure) and biological hazards.
- F.5.1 Autoclaves and Sterilization
- Autoclaves achieve higher sterilization efficacy in part because they generate wet-heat temperatures (e.g., 121°C or 250°F) higher than those achieved under standard atmospheric pressure (i.e., 100°C or 212°F). Exposure of material in an autoclave to 121°C (250°F) for 15 or more minutes is typically sufficient for sterilization, but the material’s temperature must be 121°C before the time to achieve sterilization is started. Large items, large volumes, and items that are poorly penetrated by the autoclave’s steam may take much longer than 15 minutes to sterilize. If penetration of moisture into the item is blocked, sterilization may not be achieved.
- Autoclave conditions critical to ensuring reliable sterilization methods are proper temperature and time and the complete replacement of autoclave chamber air with steam (i.e., no entrapment of air). Some autoclaves utilize a steam-activated exhaust valve that remains open during the replacement of air by live steam until the steam triggers the valve to close. Others utilize a precycle vacuum to remove air prior to steam introduction.
- Standard autoclave conditions for the types of materials that need sterilization should be established. Autoclave treatment conditions to achieve sterility will vary in relation to the volume of material treated, volume of the autoclave, the contamination level, the moisture content, and other factors. Treatment conditions for typical materials are listed below:
- Laundry: 121°C (250°F) for a minimum of 30 minutes.
- Trash: 121°C (250°F) for at least 45 minutes per bag. Size of the autoclave and size of the bags greatly affect sterilization time. Large bags in a small autoclave may require 90 minutes or more.
- Glassware: 121°C (250°F) for a minimum of 25 minutes.
- Liquids: 121°C (250°F) for 25 minutes for each gallon.
- Animals and bedding: Steam autoclaving is not recommended (sterilization time required would be at least eight hours). Incineration in an approved facility is the recommended treatment of these wastes.
- F.5.2 Autoclave Operation and Safety
- This section provides general autoclave operation information and guidelines that should be used when applicable to the operation and as needed to ensure operator safety and sterilization. In addition, specific requirements and operational procedures noted in the autoclave owner’s manual should be followed since each autoclave may have unique characteristics. The owner’s manual should be readily available to answer autoclave operational questions.
- F.5.2.1 Autoclave Instruction
- Autoclave warning and procedure posting. Source: Berkeley Lab EHS.
- The supervisor and work lead must ensure that the autoclave operator understands the autoclave hazards, controls needed to protect themselves, and any procedures necessary to accomplish sterilization for biosafety purposes.
- F.5.2.2 Autoclave Operation Modes
- Autoclaves typically use different combinations and patterns of high heat, vacuum, and pressure to sterilize the load. These combinations and patterns are used in autoclave run cycles or runs and are based on the type of material to be sterilized. General types of runs include liquids for any type of water-based solutions, dry goods with vacuum, and dry goods without vacuum. Autoclaves often have an additional drying cycle in which hot air is drawn through the chamber to dry materials after sterilization. Controls for different autoclaves vary, so the manufacturer’s instructions regarding loading, load sizes, cycle types, and settings should be carefully followed. Additional information typical of these different run cycles is listed below:
- Liquids Run. This run is longer than the other two runs, but uses lower temperatures to minimize evaporation of the liquids being sterilized.
- Dry Goods with Vacuum Run. This run moves steam and heat into the deepest parts of large bags or bundles of materials and provides the best conditions for killing resistant organisms. During this type of run, the chamber alternates between cycles of high pressure, steam, and vacuum. It is important that steam and pressure be able reach the entire load, so bag closures should be carefully loosened once they are in the autoclave.
- Dry Goods without Vacuum Run. This run pressurizes the chamber with steam for the duration of the cycle and then returns to normal. This process is used primarily for items that have been cleaned but need to be sterilized. Materials should be packed so that the heat and pressure can readily reach the whole load.
- F.5.2.3 Autoclave Container Selection
- Bags, pans, and other containers are used in the autoclave to provide primary and secondary containment for the materials and items that need to be autoclaved. Additional considerations and practices regarding these containers include:
- Polypropylene Autoclave Bags. Autoclave or biohazard bags that may be used to contain solid materials are tear-resistant but can be punctured or burst in the autoclave. These bags should therefore be placed in a rigid container during autoclaving. Bags are available in a variety of sizes, and some are printed with an indicator that changes color when processed.
- Polypropylene Containers and Pans. Polypropylene is a plastic capable of withstanding autoclaving, but it is resistant to heat transfer. Materials contained in a polypropylene pan will therefore take longer to autoclave than the same materials in a stainless steel pan. The time required to sterilize material in a polypropylene container may be reduced by removing the container’s lid, turning the container on its side, or selecting a container with the lowest sides and widest diameter that will fit in the autoclave.
- Stainless Steel Containers and Pans. Stainless steel is a good conductor of heat and is less likely to increase sterilizing time, but it is more expensive than polypropylene.
- F.5.2.4 Autoclave Preparation and Loading
- Wear long pants, closed-toe shoes, body protection such as a lab coat, gloves, and safety glasses or goggles.
- Before loading the autoclave, check inside the autoclave for any items left behind by the previous user that could pose a hazard (e.g., sharps), and then clean the drain strainer.
- Load the autoclave properly according to manufacturer’s recommendations. Typical loading practices are listed below.
- Do not autoclave items containing materials such as corrosives, solvents, volatiles, or radioactive materials that may contaminate the autoclave, create an inhalation hazard, or explode.
- Use autoclave bags and autoclavable polypropylene or stainless steel pans. Other plastics may melt.
- Load liquids as follows:
- Fill liquid containers only half full.
- Loosen caps or use vented closures so that heated and expanding liquids and vapors do not cause explosion of bottles or tubes.
- Use only borosilicate glass (e.g., PyrexTM or KimaxTM) that can withstand the high autoclave temperature.
- Use a pan with a solid bottom and walls to contain the liquid and catch spills.
- Load autoclave bags as follows:
- Put bags into pans to catch spills.
- Gather bags loosely at the top and secure the top with a large rubber band or autoclave tape. This will create an opening through which steam can penetrate. Bags are impermeable to steam and therefore should not be twisted and taped shut.
- Load dry goods such as glassware as follows:
- Check plastic materials to ensure they are compatible with the autoclave.
- Put individual glassware pieces within a heat-resistant plastic tray on a shelf or rack and not on the autoclave bottom or floor.
- Add 1/4 to 1/2 inch of water to the tray so the bottles will heat evenly.
- Leave space between items in the load to allow steam circulation.
- F.5.2.5 Autoclave Cycle and Time Selection
- Autoclave cycle selections. Source: Berkeley Lab EHS.
- Ensure the door to the autoclave is fully closed and latched, and the correct cycle and time has been selected before starting the cycle. Cycle selection should be based on the type of items and packs to be autoclaved:
- Use liquid cycle with slow exhaust when autoclaving liquids to prevent contents from boiling over.
- Use fast exhaust cycle for glassware.
- Use fast exhaust and dry cycle for wrapped items.
- Time selection should be based on the items’ sizes, volumes, insulating capacity, and other characteristics as follows:
- Take into account the size of the items to be autoclaved. Larger items with more volume take longer to autoclave. For example, a 2-liter flask containing 1 liter of liquid takes longer to sterilize than four 500 ml flasks that each contain 250 ml of liquid.
- Materials with a high insulating capacity such as animal bedding or high-sided polypropylene containers increase the time needed for the load to reach sterilizing temperatures.
- Autoclave bags containing biological waste should be autoclaved for 50 minutes to ensure decontamination.
- F.5.2.6 Removing Autoclave Loads
- Practices that should be used to prevent the operator from being injured or burned while removing the load from the autoclave include:
- Wear long pants, closed-toe shoes, body protection such as a lab coat, safety glasses or goggles, and heat-resistant gloves to open the autoclave door and remove nonliquid items from the autoclave.
- When handling large volumes of liquid, wear waterproof boots (e.g., rubber), a rubber or plastic apron that extends past the top of the boots, and sleeve protectors in addition to the clothing and personal protective equipment listed above.
- Check that the run cycle is finished and the chamber pressure is zero.
- Open the door in the following manner to prevent burns caused by steam rushing out the door: Stand behind the door, slowly open the door a crack, and keep head and hands away from the opening.
- Allow liquids to cool for 10 to 20 minutes before removing the load from the autoclave. Liquids removed too soon may boil up and out of the container and burn the operator. Let the liquids cool for an extended period (e.g., one hour) before touching the load with ungloved hands. Be sure others in the area know a heat hazard is present.
- Allow loads containing only dry glassware to cool for five minutes before removing the load from the autoclave. Then let the glassware cool for about 15 minutes before touching with ungloved hands.
- F.5.2.7 Autoclave Material Staging
- The following guidelines apply to staging materials for autoclaving and cleaning:
- Materials or equipment that will be reused and are contaminated with biohazardous material or waste should be autoclaved before being washed and stored.
- Laboratories and other areas where materials or equipment are staged for autoclaving or cleaning should have separate areas or containers for items designated as “Biohazardous – To Be Autoclaved” and “Not Biohazardous – To Be Cleaned.”
- Biohazardous materials or equipment being staged for autoclaving should be sterilized or safely confined and identified at the close of each workday. Such items should not be placed in autoclaves overnight in anticipation of autoclaving the next day.
- F.5.2.8 Burn Emergencies
- If you are burned, seek medical treatment as soon as possible. Burns to the face, third-degree burns, or burns over large areas of the body should be treated as emergencies. The LBNL emergency phone number should be called (i.e., 911). Minor burns should be treated by using first aid procedures. These procedures include immersing the burn immediately in cool water, removing clothing from the burn area, and keeping the injured area cool for at least five minutes and preferably longer. Any burns to the face or eye or any burns that blister should be seen by a physician. Regardless of the degree of severity, report the burn to your supervisor and Health Services as an occupational injury.
- F.5.3 Autoclave Maintenance and Monitoring
- Assurance is needed that the autoclave is operating properly and sterilizing the load. Assurance includes routine autoclave maintenance, monitoring autoclave conditions, and maintaining documentation.
- Maintenance described in the autoclave owner’s manual should be performed to ensure the autoclave is operating properly. This maintenance typically includes periodic maintenance performed by a qualified technician and more frequent maintenance procedures performed by the operator.
- Monitoring the sterilization process and efficacy typically includes the use of different monitoring methods including:
- Mechanical Monitoring. Mechanical monitoring, a secondary method for ensuring sterilization, involves observing and recording physical aspects of the cycle such as temperature, pressure, or time. Thermometers, pressure gauges, clocks, and logs are commonly used to observe and record the run’s physical parameters. Some autoclaves have recording devices to assist in recording run cycle conditions.
- Chemical Monitoring. Chemical monitoring uses chemical indicators that change color or physical form when an autoclave bag or pack is exposed to certain autoclave temperatures. Examples include autoclave tape and special markings on autoclave bags that are used as external indicators on the outside of the load. These indicators are typically considered process indicators since they only show that the item has been processed through the autoclave at a certain temperature, but they do not show that:
- Sterilization has been achieved or that a complete sterilization cycle has occurred.
- Temperature was achieved in the innermost parts of the load unless they are carefully placed in the load. An easy way to check interior temperature is to wrap an item such as a plastic test tube or pipette tip with autoclave tape, attach string to the item, and put the item deep into the load. Then, tape the other end of the string to the outside of the bag so that the indicator can be pulled out of the bag. Recover the indicator after the run and confirm that it has also changed color. Warning: Do not open a bag of material to place an indicator in the bag if the bag may present a hazard to the operator (e.g., bag contains Risk Group 2 material).
- Biological Monitoring. Biological monitoring (or spore testing) uses live, resistant bacterial spores on strips or in self-contained vials as biological indicators that sterilization has been achieved as demonstrated by the death of the bacterial spores. Use of appropriate biological indicators at locations throughout the autoclave is considered the best and most direct indicator of sterilization. The biological indicator most widely used for wet heat sterilization is Bacillus stearothermophilus spores. Biological indicators must be used to test the efficiency of the autoclave when the autoclave is used as the final treatment of the item prior to disposal as medical waste/biohazardous waste, or when the item will be reused and is contaminated with RG2 biological materials. In these cases, tests should be performed periodically, and test records should be maintained for three years.
Autoclave monitoring. Mechanical monitoring of cycles, temperature, pressure, and time. Chemical monitoring with autoclave tape to indicate load temperature. Biological monitoring with bacteria spores in a vial to indicate load sterilization. Source: Berkeley Lab EHS. |
- The autoclave and process should be evaluated and corrected if monitoring indicates that the autoclave run conditions were not correct, temperatures were not sufficient as shown by chemical indicators, or spores on biological indicators were not killed. Discontinue use of the autoclave if it is not working properly and post a “Do Not Use” sign. Mechanical failures need to be attended by a qualified autoclave technician. When the problem is corrected, the load should be re-autoclaved to ensure sterility.
- F.6 References
- Abedon, Stephen T., Ohio State University, supplemental lecture Web site on Control of Microbial Growth dated November 21, 1998, and Web page on Sterilization and Disinfection dated March 28, 2003
- Alfa Medical article: Sterilization – Instrumental in Patient Safety, Chris H. Miller, accessed 2010
- America Biological Safety Association (ABSA) Position Paper on the Use of Ultraviolet Lights in Biological Safety Cabinets, December 2000
- Biosafety in Microbiological and Biomedical Laboratories (BMBL), fifth edition, CDC and NIH, Appendix B: Decontamination and Disinfection
- Canadian Centre for Occupational Health and Safety (CCOHS), OSH Answers, Physical Agents, Ultraviolet Radiation Web site, February 2010
- CDC Morbidity and Mortality Week Report (MMWR), Recommendations and Reports, Guidelines for Infection Control in Dental Care Health Care Settings, Appendix A – Regulatory Framework for Disinfectants and Sterilants, December 19, 2003, 52(RR17), 62-64
- Cornell University Weill Medical College, Office of EH&S, Autoclave Safety, accessed 2010
- Cornell University Weill Medical College, Office of EH&S, Biological Safety, How long does diluted bleach last? Accessed 2013. February 6, 2003 email from Clorox to Cornell University EH&S
- Food and Drug Administration (FDA), Radiation-Emitting Products, Ultraviolet Radiation Web site, February 2010
- Lawrence Berkeley National Laboratory, Biosafety Manual, 1998
- Leadley, Sam, Pam Sojda, and N.Y. Pavilion, Using Bleach as a Germicide for Manual Washing Feeding Equipment, Cooperative Extension System Web page, November 2008.
- OSHA Standards Interpretation and Compliance Letters,” EPA-registered disinfectants for HIV/HBV,” February 28, 1997
- OSHA Instruction CPL 02-02-069, Enforcement Procedures for the Occupational Exposure to Bloodborne Pathogens, January 27, 2001. Section D (Methods of Compliance), Section 23 regarding appropriate disinfectant
- University of California at Berkeley, EH&S Fact Sheet No. 33 – Using Autoclaves Safely, February 19, 2003
- University of Maryland, Biological Safety – Autoclave Safety and Autoclaving Procedures, accessed 2010
- University of South Carolina, Autoclave Safety Policy, March 3, 2008
- Wikipedia articles: iodophor, ionizing radiation, irradiation, nonionizing radiation, surfactant, ultraviolet, ultraviolet germicidal irradiation, accessed 2010
- ____________________
- Appendix G. Biological Spills and Cleanup
- G.1 Introduction and Scope
- Hazards need to be assessed and a safe response must be implemented for each spill situation. This appendix provides general guidelines for decontamination and cleanup of various types of biological materials, including:
- Precleanup considerations
- Biological spill outside a biosafety cabinet (BSC)
- Biohazardous spill inside a BSC
- Centrifuge malfunction or spill
- Radioactive and biohazardous spill outside a BSC
- Chemical and biohazardous spill outside a BSC
- Small dead animal, nest, or droppings cleanup
- Note the following Biosafety Manual sections and guidelines for additional information related to biological spills and cleanup:
- Incident, Accident, and Emergency Response (Work Process D.10). Especially note the LBNL Emergency Guide flip chart posted in your area or online for overall response guidelines for a variety of common emergencies including biological spills and personal injury. This guide also provides both emergency and non-emergency telephone numbers.
- Decontamination, Waste, and Decommissioning (Work Process D.7). Especially note the Medical and Biohazardous Waste Generator’s Guide (PUB-3095) for disposal of medical/biohazardous waste. Also note the Guidelines for Generators to Meet HWHF Acceptance Requirements for Hazardous, Radioactive, and Mixed Wastes at Berkeley Lab (PUB-3092).
- G.2 Pre-Cleanup Considerations
- Biological spill. Source: Berkeley Lab EHS.
- Generally, you may clean a biological spill when the conditions listed below are present. If these conditions do not exist, request assistance from your supervisor or call the LBNL emergency and non-emergency telephone numbers listed in the Emergency Guide as appropriate.
- Pre-cleanup conditions:
- You understand the biological and other hazards and cleanup procedures.
- Your Job Hazards Analysis (JHA) and training sufficiently cover the work to be completed.
- There is no potential for personal exposure, injury, or environmental damage.
- The appropriate spill cleanup materials and equipment are available.
- Two people can clean up the spill thoroughly within an hour.
- G.3 Biological Spill outside a Biosafety Cabinet
- If you spilled a Risk Group 1 (RG1) material, or a small dilute amount of an RG2 material, remove any contaminated clothing, wash contaminated body areas with soap and water, and proceed to Step 6.
- If you spilled a significant amount (e.g., 100 ml or more) of an RG2 or higher material, hold your breath, leave the room immediately, and close the door.
- Warn others not to enter the contaminated area. Get help as needed and call the LBNL emergency or non-emergency phone numbers in the Emergency Guide. If you leave the area, post a sign warning others to not enter the area.
- Remove and put contaminated clothing into a container for biohazardous waste disposal or autoclaving, and thoroughly wash hands and face.
- Wait 30 minutes before re-entering the area to allow dissipation of airborne biological materials (aerosols) created by the spill. Put on personal protective equipment (PPE) before re-entering the room.
- Put on the following PPE: lab coat or gown, safety glasses, and double gloves. If the risk of the material or contamination is high, wear additional appropriate PPE such as a respirator, jumpsuit with tight-fitting wrists, or shoe covers.
- Cover the spill with paper towels or other absorbent material to prevent liquid migration and aerosol production.
- Gently pour or squirt a freshly prepared solution of 10% household bleach or other appropriate disinfectant around the edges and then into the center of the spill area until the towels are soaked with the disinfectant.
- Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and up to 20 minutes for larger volumes or RG2 materials.
- Use paper towels to wipe up the spill, working from the edges into the center of the spill. If sharps or sharp fragments such as glass might be in the spill, do not touch the spill materials with gloved hands. In this case, use a dustpan and squeegee or disposable cardboard to scoop up the spill materials and sharps.
- Clean the spill areas with paper towels soaked with disinfectant, and then with paper towels wetted with water.
- Dispose of or autoclave contaminated items. Dispose of contaminated items using biohazardous waste containers, biohazard bags, sharps containers, and other means specified in the Medical and Biohazardous Waste Generator’s Guide (PUB-3095). Reusable and autoclavable items may be decontaminated using an autoclave bag and pan in an on-site autoclave.
- Remove and dispose of PPE, or place coats in lab-coat laundry bin. Wash hands with soap and water.
- Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this program.
- G.4 Biohazardous Spill inside a Biosafety Cabinet
- This procedure assumes the spill of biohazardous material of significant quantity or risk inside a biosafety cabinet (BSC).
- Ensure the BSC is operating and continues to operate during this procedure so as to prevent airborne contaminants from escaping the cabinet.
- Put on the following PPE: lab coat or gown, safety glasses, and chemical-resistant double gloves. Wear additional PPE (e.g., respirator or goggles) as needed based on the risk of the material, contamination, or splashing.
- Spray or wipe walls, work surfaces, and equipment with a disinfectant that is effective against the agents that may be present. A 1% solution of an iodophor decontaminant (Wescodyne or equivalent) is effective against most viruses, fungi, vegetative bacteria, and most nonencysted amoeba. A decontaminant detergent has the advantage of detergent activity, which is important because extraneous organic substances frequently interfere with the reaction between microorganisms and the active agent of the decontaminant.
- Flood the BSC’s top work-surface tray with disinfectant. In a Class II BSC, also flood with disinfectant the drain pans and catch basins below the work surface. Allow the disinfectant to stand for 10 to15 minutes.
- Remove excess disinfectant from the tray by wiping with a sponge or cloth soaked in disinfectant.In a Class II BSC, drain the BSC’s top work surface into the BSC catch basin, lift out the work surface and removable exhaust grilles, and wipe the top and bottom (underside) surfaces with a sponge or cloth soaked in disinfectant.Replace the work surface and grilles. Drain the disinfectant from the BSC base into an appropriate container. Place the container with disinfectant, gloves, cloth, or sponge in an autoclave pan, and then autoclave according to standard procedures.
- Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this manual.
- G.5 Centrifuge Malfunction or Spill
- This procedure assumes that the following types of centrifuge events have occurred, especially if RG2 materials are involved: the spill of biological material in the centrifuge, significant mechanical failure (e.g., rotor failure), or centrifuge tube or container breakage. Evidence of such conditions might include noises during centrifuge operation or visual signs of failure or leakage when the centrifuge is opened. Note that breakage of tubes and leakage of fluid into the centrifuge wells or cups during centrifugation may release relatively few agents into the air. However, if a tube breaks and leaks in the centrifuge chamber, then aerosols and droplets may be created and dispersed.
- In the event of a centrifuge malfunction or spill, follow the following steps:
- Turn centrifuge off immediately. Keep the centrifuge lid closed and latched.
- Notify others.
- Evacuate the laboratory if hazardous aerosols may have been generated. Close the door, post a biohazard spill sign at the lab door, and stay out of the laboratory for 30 minutes.
- For spill cleanup, the operator should wear PPE (i.e., gloves, lab coat, eye protection), remove debris, and clean and disinfect centrifuge interior, rotors, safety cups, or buckets in accordance with the manufacturer’s instructions.
- Place any contaminated PPE and all cleanup materials in a biohazardous waste container. Wash hands and any exposed skin surfaces with soap and water.
- Report spill, exposure, and injury incidents to your work lead or supervisor in accordance with Work Process D.10 of this manual.
- G.6 Radioactive and Biohazardous Spill
- This procedure assumes the spill of material outside a biosafety cabinet that has both radioactive and biohazardous concerns. In this case, the biological component of the spill should be inactivated prior to disposal of the spilled materials as radioactive waste. Call the Radiation Protection Group at extension 7277 or (510) 486-7277 for instruction and assistance.
- Spill of RG1 Material or Small amount (e.g., Less Than 100 ml) of Dilute RG2 Material
- Warn others not to enter the contaminated area. Post a sign on the door as needed.
- Remove any contaminated PPE (e.g., lab coat, gloves) if there is a risk of exposure to biohazardous agents, and isolate PPE in a plastic bag or appropriate container.
- Contact the Radiation Protection Group (RPG) 24/7 at extension 7277 or (510) 486-7277 to report the incident. If RPG is responding to the spill location, wait until RPG arrives before proceeding with the steps below.
- Monitor yourself for radioactive contamination. If contaminated,wait for RPG assistance.
- Thoroughly wash your hands and face if there is a risk of exposure to biohazardous agents.
- Put on the following PPE: lab coat or gown, safety glasses, and double gloves. If the risk of the material or contamination is high, wear additional appropriate PPE such as respirator, jumpsuit with tight-fitting wrists, or shoe covers.
- Cover the spill with paper towels or other absorbent material to prevent liquid migration and aerosol production.
- Gently pour or squirt a freshly prepared solution of 10% household bleach or other appropriate disinfectant around the edges and then into the center of the spill area until the towels are soaked with the disinfectant.
- Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and up to 20 minutes for larger volumes or RG2 materials.
- Use paper towels to wipe up the spill, working from the edges into the center of the spill. If sharps or sharp fragments such as glass might be in the spill, do not touch the spill materials with gloved hands. In this case, use a dustpan and squeegee or disposable cardboard to scoop up the spill materials and sharps.
- Clean the spill areas with paper towels soaked with disinfectant, and then with paper towels wetted with water.
- Place all contaminated materials into a plastic bag, and place the bag in the appropriate radiation waste container. Monitor for radiation contamination all potentially contaminated items that are not placed in the radiation waste container. Decontaminate and resurvey these items as necessary.
- Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this program.
- Spill of Risk Group 2 Material Greater Than 100 ml
- If you spilled a significant amount (e.g., 100 ml or more) of an RG2 material, hold your breath, leave the room immediately, and close the door.
- Warn others not to enter the contaminated area.
- If possible, remain stationary and request assistance from others to contact the Radiation Protection Group (RPG). Contact RPG 24/7 at extension 7277 or (510) 486-7277 for assistance.
- Remove any contaminated PPE (e.g., lab coat, gloves) if there is a risk of exposure to biohazardous agents, and isolate PPE in a plastic bag or appropriate container.
- Thoroughly wash your hands and face if there is a risk of exposure to biohazardous agents.
- Proceed with the remaining steps after RPG’s arrival.
- Wait 30 minutes before re-entering the area to allow dissipation of airborne biological materials (aerosols) created by the spill. Put on PPE before re-entering the room.
- Follow Steps 6 through 13 noted in the previous section titled “Spill of RG1 Material or Small Amount (e.g., Less Than 100 ml) of Dilute RG2 Material.”
- G.7 Chemical and Biohazardous Spill
- This procedure assumes that the spill of a material took place outside a biosafety cabinet, the material has both chemical and biological hazards, the chemical in the material is considered a hazardous waste, and the chemical has not already rendered the biological material nonviable or inactive.
- Prior to starting your research, determine which chemical disinfectant(s) and absorbent materials are compatible with the chemical(s) that may become biologically contaminated and whether the contaminated chemical(s) can be autoclaved.Autoclaves heat materials at high temperatures and pressures, and the autoclave operator may be exposed to chemical vapors when the autoclave is opened.
- If you spilled a significant amount (e.g., 100 ml or more) of an RG2 material, hold your breath, leave the room immediately, and close the door.
- Warn others not to enter the contaminated area. Get help as needed. If you leave the area, post a sign warning others to not enter the area.
- Remove and put contaminated clothing in container lined with a plastic bag for eventual decontamination, autoclaving, or disposal. Thoroughly wash hands and face. If clothing is chemically contaminated, autoclaving may not be advisable.
- If you evacuated the laboratory as stated in Step 2, call the LBNL emergency or non-emergency phone numbers in the Emergency Response Guide and wait 30 minutes before re-entering the area to allow dissipation of airborne biological materials (aerosols) created by the spill. Put on PPE before re-entering the room.
- Consult the LBNL Chemical Hygiene and Safety Plan for chemical spill response procedures. If the chemical(s) in the spill present a greater hazard than the biological agent(s), proceed with chemical decontamination first.
- Put on at least the following PPE: lab coat or gown, safety glasses, and chemical-resistant double gloves. If the risk of the material or contamination is high, wear additional appropriate PPE such as a respirator, a jumpsuit with tight-fitting wrists, or shoe covers.
- Cover the spill with an absorbent material or towel that will not react chemically with the spilled chemical. Towels will prevent liquid migration and aerosol production.
- Use a disinfectant that is compatible with the chemical(s) in the spill. Gently pour or squirt the disinfectant around the edges and then into the center of the spill area until the absorbent material or towel is soaked with the disinfectant.
- Let the disinfectant stay in contact with the spilled material for at least 10 minutes, and up to 20 minutes for larger volumes or RG2 materials.
- Use chemically compatible towels, dustpan, squeeges, or cardboard to scoop and wipe up the spill, working from the edges into the center of the spill. If there might be sharps or sharp fragments such as glass in the spill, do not touch the spill materials with gloved hands.
- Clean the spill areas with towels soaked with disinfectant, and then with towels wetted with water.
- If the chemical(s) are compatible with autoclaving, contaminated materials (paper towels, absorbent, glass, liquid, gloves, dustpan, squeegee, etc.) may also be placed into autoclave bags and an autoclave pan. Cover the pan with aluminum foil and autoclave according to standard directions. After autoclaving, the now-sterile materials may require being disposed of as hazardous chemical waste via the LBNL Waste Management Group.
- If the chemical(s) are not autoclavable (or if you do not know whether they are autoclavable), then transfer the disinfected materials into a screw-cap container, and place the container in the Satellite Accumulation Area.
- Report spill, exposure, and injury incidents to your work lead or supervisor and in accordance with Work Process D.10 of this section.
- G.8 Cleanup of Small Dead Animals, Nests, or Droppings
- The following general procedure should be used as a guideline for cleanup of small dead animals, nests, or droppings. This procedure may need to be adapted depending on the nature of the materials and situation. Contact the Facilities Division via the Facilities Work Request Center if assistance is needed due to a pest infestation or to the nature or size of the concern.
- Wear PPE such as reusable or disposable rubber gloves and safety glasses when handling decontaminant solutions, dead animals, or cleaning up contaminated materials. Use double disposable gloves if possible and appropriate. Determine if disposable or cleanable protective clothing is also needed.
- Clean up dead animals, nests, droppings or contaminated food by first spraying or soaking the item with an appropriate disinfectant such as 10% household bleach, Lysol®, or other appropriate janitorial disinfectant (see Appendix F, Section F.2.3 of this manual). Allow the disinfectant sufficient time to decontaminate the item (e.g., 10 minutes).
- If possible and appropriate, pick up the decontaminated item with an impervious barrier such as a plastic bag placed over the item. Place the decontaminated item into a plastic bag, tie the bag shut, place the bag into a second bag, and tie the second bag shut.
- Clean up localized gross surface contamination as needed by spraying or soaking with disinfectant and using disposable paper towels. Place waste materials in a plastic bag, remove outer contaminated disposable gloves, and double bag the waste materials.
- Dispose of the bags of waste in the general trash. Use an outside dumpster as needed to prevent odor problems.
- Clean contaminated surfaces or floors as needed. Use a solution of water, detergent, and disinfectant to mop floors or wipe surfaces. Steam clean or shampoo carpets and upholstered furniture. Do not vacuum or dry sweep surfaces before wet cleaning. Pour mop or cleaning wastewater into a drain that is connected to the building’s sanitary sewer system.
- Remove PPE, and then clean it or dispose of it.
- Remove any potentially contaminated clothing and launder separately with detergent and hot water.
- Wash hands with soap and water.
- The state of California encourages the reporting of dead birds and squirrels to assist state agencies in tracking disease. This reporting is optional at LBNL and involves keeping the animal for 24 hours without decontamination or freezing. See the California West Nile Virus Web site for additional information and online reporting.
- ____________________
- Appendix H. Transportation and Shipping
- H.1 Introduction and Scope
- This appendix provides requirements, guidelines, and direction on transporting and shipping biological materials as needed to safely move the material from one location to another. This includes:
- Employee transport of biological materials between laboratories, between buildings, in motor vehicles, and on LBNL buses
- Use of LBNL Receiving, Transportation, and Shipping
- Shipping through LBNL Shipping by a contracted shipping company (e.g., common carrier such as FedEx or UPS)
- Packaging, transportation, and shipping in accordance with:
- U.S. Department of Transportation (DOT) Hazardous Materials Regulations (HMR) for movement of biological materials in public right-of-ways within the U.S.
- International Air Transport Association (IATA) Dangerous Goods Regulations (DGR) for shipment of biological materials (e.g., infectious substances) by air.
|
- Employees who wish to transport or ship a biological material should use this appendix (starting in Section H.2) to assess if the material is a regulated biological material and select a mode and process for moving the material. Modes and processes detailed in this appendix cover safe movement of all biological materials and potential shipping and transportation regulatory issues, although most LBNL biological materials that need to be moved are not regulated. Regulatory requirements for packaging, transporting, and shipping are applicable only if the material is:
- Moved in vehicles, airplanes, railcars, or vessels via public right-of-ways such as roadways, airways, railways, and sea lanes that are accessible to the public and
- A regulated biological material (i.e., categorized by DOT HMR or IATA DGR as an infectious substance or genetically modified organism).
- This appendix does not cover the following topics:
- Transportation and shipping of nonbiological hazardous materials. These topics are covered in the following LBNL documents:
- The ESH Manual Transporting and Shipping Hazardous Materials program:
- The Chemical Hygiene and Safety Plan (CHSP), “Chemical Procurement, Transportation, and Inventory”
- Other regulatory requirements related to the import, export, and transfer of biological materials. See Appendix I of this manual for information on these topics.
- H.2 How to Determine Transportation Mode and Requirements
- LBNL employees should use the following steps to determine the transportation mode and requirements needed to transport or ship a biological material:
- Determine the desired mode of transportation or shipping.
- Use Table H-1 to determine whether the desired transportation mode can be used. If needed, use Section H.4 to determine whether the material is subject to IATA or DOT shipping regulations. Section H.4 can also be used for definitions of terms.
- Use Section H.3 to determine the requirements or process for packaging, labeling, transporting, or shipping the material.
- Table H-1. Transportation Modes and Biological Materials Not Allowed
General |
Specific |
Biological Materials That Are |
Personal Transportation
|
Hand carry between laboratories |
No restrictions on types of biological materials |
Hand carry between buildings |
No restrictions on types of biological materials |
|
Personal motor vehicle* |
Regulated** biological materials are not allowed except for regulated materials contained in human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, cells, and body parts) being transported for research, diagnosis, investigational activities, or disease treatment or prevention; or that are biological products. Samples containing Category A infectious substances are not allowed. |
|
LBNL shuttle bus or other public transportation |
Regulated** biological materials or other biological materials that may present a detrimental risk to the health of humans or other organisms either directly through infection or indirectly through damage to the environment are not allowed. |
|
Licensed Transporter
|
LBNL Transportation Department |
No restrictions on types of biological materials. |
Common carrier |
No restrictions on types of biological materials unless restricted by the carrier. |
Footnotes: * Personal transport in a motor vehicle means transportation in a private or government passenger vehicle such as a car, van, or pickup truck. **Materials that are and are not subject to DOT and IATA regulations are described in Section H.4 of this appendix. |
- Here is an example of how to apply Steps 1, 2, and 3 above:
- An LBNL research employee wants to transport his established human cells in a personal vehicle between two LBNL sites in direct support of his research project. According to Table H-1, this is allowable because it is a human sample being transported solely for the purpose of research, regardless of whether or not the human cells are a regulated biological material. According to the second bullet in Section H.4.1, these cells would not be considered regulated biological materials unless they contained infectious agents or were collected from individuals suspected of having an infectious disease; however, this determination does not matter, because this is a human sample being transported in direct support of a research project. The researcher must package and label the human cells according to Section H.3.1.3, Personal Transport in Motor Vehicle. The researcher may then give the packaged cells to another person affiliated with the research for transport in a personal vehicle if this individual is affiliated with the research, knows the cells are in the vehicle, is informed of the applicable requirements in this appendix, and is doing the transport solely for the purpose of supporting the research.
- H.3 Requirements and Processes for Receiving, Transporting, and Shipping
- This section presents requirements and processes related to receiving, transporting, and shipping biological materials by an LBNL employee, LBNL Transportation or Shipping Groups, or a common carrier. See Section H.2 to determine whether the desired mode of transportation or shipping can be used to transport the biological material.
- H.3.1 Employee Transportation of Materials
- This section covers minimum requirements for transporting biological materials by an LBNL employee without the use of the LBNL Transportation Group or a common carrier. The following are objectives that should be accomplished whenever employees transport biological materials:
- Biological materials will not be spilled in the event of an accident (e.g., due to a person tripping or a vehicle accident).
- The identity of biological materials, their hazards or lack of hazards, and owners can be explained by people transporting the materials and determined by other people who may find the materials.
- Exterior surfaces of containers will not be contaminated with biological materials.
- Regulated biological materials being transported in public right-of-ways (e.g., in vehicles on roads or in airplanes) will be packaged and transported in accordance with DOT and IATA regulations.
- H.3.1.1 Hand-Carry Transport between Laboratories
- Primary container example
- Secondary container used to transport materials between laboratories. Source: Berkeley Lab EHS.
- Containers inside a break-resistant and leak-proof carrier. Source: VWR (2010).
- Hand-carry transport between laboratories generally means an LBNL employee is hand-carrying the biological material in a container and walking between laboratories in the same building or buildings that are closely connected and designed for pedestrian traffic. Requirements and precautions for such transport include:
- Primary or secondary containers that prevent leakage are required. When Risk Group (RG) 2 or bloodborne pathogen (BBP) materials are transported, a biohazard label must be displayed on the exterior of the outermost container. When possible and appropriate for the work and risk:
- Primary containers of biological material should be break-resistant (e.g., plastic), leak-proof, have secure caps or lids, and be disinfected on the outside.
- Primary containers of biological material should be placed in a secondary container that prevents leakage. Racks or packing should be used inside the secondary container as needed to keep the primary containers upright and prevent breakage.
- The primary or secondary containers should be labeled with the identity of the contents, ownership information, and any appropriate biohazard information. Such labeling may not be needed if the primary container(s) and secondary container will remain in continuous possession of the person(s) transporting and processing the materials.
- Medical/biohazardous waste must be transported in accordance with the container and labeling requirements in the Medical and Biohazardous Waste Generator’s Guide (PUB-3095).
- H.3.1.2 Hand-Carry Transport between Buildings
- Hand-carry transport between buildings generally means the packaged biological material is carried by an LBNL employee who is walking between nonadjacent LBNL or University of California, Berkeley (UCB) buildings. Requirements and precautions for such transport include:
- Biological materials transported by this means are not subject to DOT and IATA regulations, but the biological materials should be transported according to the packaging and labeling criteria described in Section H.3.1.3, Personal Transportation in Motor Vehicle of this appendix.
- Employees transporting materials by this means should take precautions to ensure they can walk safely between buildings. Precautions may include having one hand free to open doors and hold stair rails, use of a hand truck, and wearing slip-resistant shoes.
- Medical/biohazardous waste cannot be transported off LBNL sites (e.g., between discontinuous LBNL locations or different institutions). Medical/biohazardous waste must be transported in accordance with the container and labeling requirements in the Medical and Biohazardous Waste Generator’s Guide (PUB-3095).
- H.3.1.3 Personal Transportation in a Motor Vehicle
- Personal transportation in a motor vehicle means transportation by an LBNL employee in a private or government passenger vehicle such as a car, van, or pickup truck. Requirements for such transport of biological materials are described in this section. These requirements meet the DOT HMR requirements for transporting materials of trade:
- Materials allowed. Materials that may be transported in a motor vehicle include unregulated biological materials noted in Section H.4.1, the regulated materials noted as an exception in Table H-1, and dry ice. Other regulated biological materials or medical/biohazardous waste are not allowed. Transportation of any regulated biological material must be in direct support of a principal business (e.g., research project), and the principal business must not be motor vehicle transportation (e.g., a company paid to transport items).
- Packaging and labeling. An inner container and outer package are required.
- Manufacturer’s packaging. When applicable, each regulated biological material must be contained and packaged in the manufacturer’s original container and packaging, or a container and packaging of equal or greater strength and integrity.
- Inner containers:
- Use break-resistant (e.g., plastic) containers, if possible.
- Liquids must be in a leak-proof container. Lids on inner containers must have a positive means of closure. For example, a screw cap should be used instead of parafilm, aluminum foil, or a stopper.
- Container(s) must be disinfected as needed for safety and should be placed in a Ziploc® bag or an equivalent secondary spill container.
- Information must be placed on or with the container(s) as needed to clearly communicate the container’s contents, hazards, and ownership. Each individual container must be labeled with enough information to identify its contents. In addition, the container(s) or secondary bag(s) must also be labeled with the identity of the material, the name and phone number of the sender, the recipient’s name and phone number if they are different from the sender’s, and hazard information. Hazard information includes a biohazard label if the material is biohazardous (e.g., RG2), any words needed to explain the hazard, or words indicating the material is not hazardous.
- Containers for sharps (i.e., sharps container) must be constructed of a rigid material resistant to punctures and securely closed to prevent leaks or punctures.
- Example of packaging and labeling of unregulated biological materials for
- transportation in a motor vehicle. See text for criteria and requirements.
- Source: Berkeley Lab EHS.
- Outer packaging:
Ice chest with secured lid used as an outer package. Source: unidentified.
- The outer packaging must be a strong and tight packaging made of a rigid material. It must also be securely closed. Examples include a cardboard, plastic, or metal box or pail with a secure lid. A plastic carrier that is leak-proof, easy to clean, and has a secure lid is typically the best package for biological materials (e.g., ice chest or enclosed laboratory tube carrier).
- Packing material or racks must be used between the inner container(s) and outer packaging as needed to keep the container(s) upright, cushion the container(s), and prevent the container(s) from shifting or damage.
- Sufficient absorbent material must be inside the outer packaging to absorb the entire contents of all inner liquid container(s).
- The exterior of the outer packaging must be labeled with the same information required for the inner container. The common name(s) or shipping name(s) of the materials must be used.
- Outer packaging must be secured against shifting inside the vehicle during transport. Generally, the safest place to secure biological materials is in a vehicle trunk. If hazardous materials are also transported, these materials must be placed in the trunk or truck bed.
- Material quantity of regulated biological material:
- Each inner container must not be more than 0.5 kg (1.1 lbs) or 0.5 L (17 ounces), and an aggregate contained within the entire outer package must not be more than 4 kg (8.8 lbs) or 4 L (1 gallon) or
- A single inner container containing not more than 16 kg (35.2 lbs) or 16 L (4.2 gallons) that is inside a single outer package.
- Ice and dry Ice. Ice and dry ice may be used inside the package to keep the biological materials cold. Ice must be packaged so that any melting water will be contained inside the outer packaging. Dry ice is frozen carbon dioxide that will sublimate into gas, so dry ice must be placed in packaging that is not gas-tight (e.g., ice chest). Dry ice is only regulated as a hazardous material in air transport, but is not regulated in ground (e.g., motor vehicle) transport in the U.S.
- Hazard communication. The operator of a motor vehicle that contains a regulated biological material must be informed of the presence of the material, and must be informed of the requirements in this section.
- H.3.1.4 Personal Transportation on an LBNL Shuttle Bus
- Personal transportation on an LBNL shuttle bus means the packaged biological material is carried by an LBNL employee on an LBNL shuttle bus. The following materials must not be transported on an LBNL bus: regulated biological materials, medical/biohazardous waste, or other biological materials that may present a detrimental risk to the health of humans or other organisms, either directly through infection or indirectly through damage to the environment. Any other biological materials transported by this means are not subject to transportation regulations, but the biological materials should be transported according to the packaging and labeling criteria described in Section H.3.1.3, Personal Transportation in a Motor Vehicle, above.
- H.3.2 LBNL Receiving, Transportation, and Shipping
- Receiving, transportation, and shipping of biological materials are conducted institutionally from Building 69 by Shipping/Receiving/Transportation within the Facilities Division. These services are conducted in accordance with ESH Manual Transporting and Shipping Hazardous Materials program, DOT HMR, IATA DGR, and by personnel with appropriate regulatory qualifications. For questions about shipping or receiving biological materials, contact LBNL Shipping at (510) 486-5084 or LBNL Receiving at (510) 486-4935.
- H.3.2.1 LBNL Receiving
- Biological materials that are shipped by a contracted shipping company (i.e., common carrier) to LBNL must be received by LBNL Receiving and are typically delivered to the requestor via LBNL Transportation in the packaging and with the documentation that was received from the common carrier.
- H.3.2.2 LBNL Transportation
- This section covers the pickup and delivery of biological materials or items that contain biological materials (e.g., freezers) within LBNL by LBNL Transportation or a carrier authorized by Transportation. Transportation of materials must be requested through the Facilities Work Request Center, and a completed Transportation Authorization Form (TAF) must be attached to each item to be transported. Additional directions include:
- When placing a work request for transportation, the requestor will be asked if the item to be transported contains hazardous materials. The requestor should declare that the item does not contain hazardous materials if the item to be transported does not contain regulated biological material as described in Section H.4 or other hazardous materials.
- If the item does not contain a regulated biological or other hazardous material, the requestor should package and label the biological materials as described in Section H.3.1.3, Personal Transportation in Motor Vehicle, of this appendix.
- If the item contains a regulated biological or other hazardous material, the requestor should consider personal transportation of the item in a motor vehicle (see Section H.3.1.3 of this appendix) if allowed (see Table H-1), or contact LBNL Shipping for advice and directions.
- See Section H.3.2.3 below if the item will also be shipped by a common carrier after transportation within LBNL.
- H.3.2.3 LBNL and Common Carrier Shipping
- Trained person in LBNL Shipping ensures the material is correctly packaged, labeled, and documented. Source: Berkeley Lab EHS.
- Shipment of biological materials by a common carrier out of LBNL must be conducted by LBNL Shipping. Information and assistance must be provided by the sender. Use the following guidelines for shipping:
- Note directions for transportation and pickup of materials in Section H.3.2.2.
- An LBNL Shipping Document must also accompany all material leaving LBNL. Directions for completing this form can be found on the LBNL Shipping Web site. This form requires the sender to describe the item and material to be shipped, and asks if the item and material is a regulated hazardous material (i.e., contains dangerous goods).
- The sender may use the lists of unregulated and regulated materials in Section H.4 to answer the Dangerous Goods question on the form in regards to biological materials. Section H.4 can also be used to determine what information should be included in the form’s description section. The sender is responsible for providing a description of the item and biological material and its potential biological or hazardous materials risks so that LBNL Shipping can correctly categorize and ship the material.
- Trained personnel in LBNL Shipping determine whether the material is subject to DOT and IATA shipping regulations. They also ensure the material is correctly packaged, labeled, and documented for shipment. If the material is a regulated biological material, LBNL Shipping will work with the sender to ensure the shipping requirements are implemented at the sender’s LBNL location.
Packaging and labeling as an infectious substance. Transporting Infectious Substances Safely, US DOT Document PHH50-0079-0706 (October 1, 2006). |
- H.4 Unregulated and Regulated Materials
- This section provides information on which biological materials are or are not subject to DOT HMR and IATA DGR infectious substance and genetically modified organism shipping regulations. LBNL employees should use this information to assist in selecting or requesting appropriate modes of transport for their biological materials.
- H.4.1 Unregulated Biological Materials
- The following materials are not subject to DOT and IATA infectious substance shipping regulations:
- Substances that do not contain infectious substances or that are unlikely to cause disease in humans or animals.
- Noninfectious biological materials from humans, animals, or plants. Examples include noninfectious cells, tissue culture, blood, or plasma from individuals not suspected of having an infectious disease, DNA, RNA, or other genetic elements.
- Substances containing microorganisms that are nonpathogenic to humans or animals.
- Substances that have been neutralized or inactivated so that they no longer pose a health risk.
- Environmental samples that are not considered to pose a significant risk of infection (e.g., food and water samples).
- Dried blood spots.
- Fecal occult blood screening tests.
- An infectious substance (other than a Category A infectious substance) contained in a patient sample being transported for research, diagnosis, investigational activities, or disease treatment and prevention; or a biological product when such materials are being transported by a private carrier in a motor vehicle used exclusively to transport such materials.
- Blood or blood components that have been collected for the purpose of transfusion or the preparation of blood products to be used for transfusion or transplantation.
- Tissues or organs intended for use in transplantation.
- A material with a low probability of containing an infectious disease, or where the concentration of the infectious substance is at a level that naturally occurs in the environment and cannot cause disease when exposure to it occurs. Examples of these materials include foodstuffs and environmental samples (e.g., samples of water, dust, or mold).
- A biological product, including an experimental or investigational product or component of a product, subject to federal approval, permit, review, or licensing requirements such as those required by the Food and Drug Administration (FDA) or U.S. Department of Agriculture (USDA).
- H.4.2 Regulated Biological Materials
- The materials presented below are subject to DOT and IATA shipping regulations for infectious substances and genetically modified organisms:
- Infectious substances are materials regulated for shipping. These materials are known to be, or are reasonably suspected to contain, an animal or human pathogen. A pathogen is a virus, microorganism (including bacteria, plasmids, or other genetic elements), proteinaceous infectious particle (prion), or a recombinant microorganism (hybrid or mutant) that is known or reasonably expected to cause disease in humans or animals. Microorganisms that are unlikely to cause human or animal diseases are not subject to biological shipping regulations.
- Category A infectious substances are materials capable of causing permanent disability, or a life threatening or fatal disease in humans or animals when exposure to them occurs. Category A infectious substances are shipped as infectious substances affecting humans (UN2814) or infectious substances affecting animals (UN2900). Examples of Category A infectious substances are listed in a table in the infectious substances section of the IATA Dangerous Goods Regulations.
- Category B infectious substances are materials that do not meet Category A criteria. Category B infectious substances are shipped as UN3373.
- Patient specimens or diagnostic specimens are any human or animal materials including but not limited to excreta, secreta, blood, blood components, tissue, and tissue fluids being shipped for the purpose of diagnosis. Patient specimens that have a minimal likelihood of containing pathogens are regulated materials, but they are also exempt from many shipping requirements. Professional judgment is used to determine if a specimen contains pathogens and should be based on the patient’s medical history, symptoms, local conditions, and individual circumstances. The outer package must be marked “Exempt human specimen” or “Exempt animal specimen.” If there is more than a “minimal likelihood” that a patient specimen contains pathogens, it must be shipped as a Category A or Category B infectious substance.
- Biological products are materials that are derived from living organisms and manufactured for use in the prevention, diagnosis, treatment, or cure of disease in humans or animals and are certified by the USDA, FDA, or other national authority. Examples of biological products include certain viruses, therapeutic serums, toxins, antitoxins, vaccines, blood, and blood products. Biological products transported for final packaging, distribution, or use by medical professionals are not subject to biological shipping regulations. Biological products that do not meet these criteria must be shipped as UN2814, UN2900, or UN3373 when appropriate.
- Genetically Modified Organisms (GMO) or microorganisms (GMMO) are organisms whose genetic material has been purposely altered through genetic engineering in a way that does not occur naturally. GMOs and GMMOs that do not meet the definition of toxic or infectious substances but can alter animals, plants, or microorganisms in a way that is not normally the result of natural reproduction are considered a miscellaneous hazard (Class 9) and must be shipped as UN3245. GMOs and GMMOs that are infectious must be shipped as UN2814, UN2900, or UN3373.
- H.5 References and Resources
- International Air Transport Association (IATA) Dangerous Goods Regulations (DGR), Section 3.6.2, “Division 6.2 : Infectious Substances,” and Section 3.9, “Class 9: Miscellaneous Dangerous Goods, Genetically Modified Microorganisms and Genetically Modified Organisms”
- PUB-3095, Medical and Biohazardous Waste Generator Guidelines, LBNL, latest revision
- Transporting Infectious Substances Safely, guide to changes effective October 1, 2006, US DOT Document PHH50-0079-0706
- UNH Shipment of Biological Materials Manual, University of New Hampshire, 2011
- UNH Guide to Shipping with Dry Ice, April 9, 2007
- U.S. Department of Transportation (DOT) Hazardous Material Regulations (HMR): 49 CFR 171.8 (Definitions), 49 CFR 173.134 (Infectious Substances), and 49 CFR 173.6 (Materials of Trade)
- U.S. Postal Service (USPS) Domestic Mail Manual Section 10.17, Infectious Substances
- ____________________
- Appendix I. Import, Export, and Transfer Restrictions
- I.1 Introduction and Scope
- Materials being transferred (i.e., imported, exported, or transferred) from one location or person to another may be subject to regulatory restrictions or permit requirements. United States (U.S.), state, and foreign government agencies restrict and permit the movement of certain biological materials across borders to prevent threats to public health, agriculture, environment, and national security.
- This appendix provides an outline of U.S.-based regulatory restrictions, permits, and lists related to the transfer (i.e., import, export, or transfer) of biological and related materials. This outline provides LBNL personnel with a starting point for determining whether such materials are potentially regulated by U.S. agencies, and whether there are restrictions or permits applicable to transfer of the material or equipment. Contact the LBNL Biosafety Office (biosafety@lbl.gov) for additional advice.
- This appendix does not provide comprehensive information about restricted materials, or transfer or shipping requirements. Additional LBNL policy information may be found in the following documents:
- Web links and references to external agencies provided in this appendix
- Appendix H of this manual for transportation and shipping requirements
- The Berkeley Lab Export Control Manual for general LBNL export control requirements
- The supervisor, work lead, person transferring the biological material, person requesting transfer of the biological material, and permit holder all have LBNL or legal responsibilities for complying with transfer requirements, obtaining any required permits, and following the conditions of the permit. Regulatory requirements, permits, and permit conditions related to the transfer of biological materials should also be included in the Biosafety Work Authorization. The LBNL Biosafety Office and Institutional Biosafety Committee (IBC) will review the researcher’s assessment and documentation of transfer requirements during the work authorization review process.
- I.2 Importing or Transfer into the U.S. and California
- There may be restrictions or permits required for the transfer of biological material between collaborators, or for importing material into the U.S. from foreign countries or in some cases into California or the San Francisco Bay Area.
- USDA-APHIS label for shipping soil samples under a soil permit.
Source: Berkeley Lab EHS. - Shipments and persons entering the U.S. are processed by the U.S. Customs and Border Protection (CBP), which is a branch of the Department of Homeland Security. The CBP checks materials transported by travelers and shipments for proper import permits, packaging, and labeling. This check may include opening and inspecting the package. Noted concerns may be reported to other U.S. agencies. In addition, the California Department of Food and Agriculture (CDFA) and the U.S. Department of Agriculture – Animal and Plant Health Inspection Service (USDA-APHIS) do not allow the import of certain materials that may be infested with invasive species identified as pests by the state. CDFA also has border protection stations that inspect vehicles for commodities that may be infested with pests. The person importing the material (the importer) should therefore:
- Obtain an import permit from the appropriate government organization prior to shipment, if required
- Package and label the material according to permit and shipping requirements
- Consider including a courtesy letter (e.g., a letter that describes the contents in detail and any hazards, concerns, permit requirements, or lack thereof) with the shipment
- Prior to shipment of the material, the person importing the material (the importer) should contact the appropriate government organization to determine its transfer requirements. The importer is legally responsible for ensuring that personnel package, label, and ship regulated material from the foreign country according to the regulating agency’s requirements and shipping regulations. Shipping labels are often also issued to the importer with the permit. The importer must send the labels and one or more copies of the permit to the shipper. The permit and labels inform CBP and other agencies of the package contents.
- I.2.1 CDC and APHIS Select Agent and Toxin Restrictions
- Select agents and toxins are specific pathogenic agents and toxins that pose a severe threat to human, animal, and plant health because of their potential for use as biological weapons. They are therefore regulated by the Department of Health and Human Services – Centers for Disease Control and Prevention (HHS-CDC) and USDA-APHIS. See Work Process B.3.d.v of this program for additional information; and Appendix B, Section B.2 and Section B.3, for a list of select agents and toxins. Consult the most recent online list at www.selectagents.gov.
- Only facilities registered with and individuals approved by CDC or APHIS are allowed to possess, have access to, or transfer the specific agents and strains or toxins for which they are approved. Transfers of select agents or toxins must be conducted with the approval and involvement of the LBNL EHS Biosafety Office.
- I.2.2 APHIS Agricultural Permits
- The USDA-APHIS defends America’s animal and plant resources from agricultural pests and diseases by regulating materials, organisms, or agents that may harm domestic or native animals or plants, or natural resources. These materials, organisms, or agents may cause harm directly (e.g., predator or pathogen) or indirectly (e.g., vector). Generally, APHIS requires a permit or another document issued to an individual to import, export, or store regulated materials from or to locations outside the continental U.S. or between U.S. states.
- Work Process B.3.e, USDA-Regulated Materials, Organisms, and Agents, of this program provides an overview of APHIS agency branches along with categories and examples of regulated materials, organisms, and agents. The following sections provide additional agency details, requirements, and Web links for more information.
- I.2.2.1 APHIS Plant Health Permits
- The Plant Protection and Quarantine (PPQ) branch of APHIS safeguards agriculture and natural resources from the risks associated with the entry, establishment, or spread of animal and plant pests and noxious weeds to ensure an abundant, high-quality, and varied food supply. PPQ provides the following resources:
- PPQ Permits Web page: Provides permit applications for soil, plant pests, plants, plant products, weeds, etc.
- The PPQ Soil Circular: Defines what is and is not soil, and provides information about soil treatments and permits. Soil is a mixture of inorganic and organic materials, when the organic materials are unidentifiable plant and/or animal parts. This mixture can support biological activity and therefore carry and introduce harmful pests or diseases from one location to another.
- The PPQ Plant Pest Program: Provides a list of select insects, mollusks, nematodes, plant diseases, or noxious weeds that are considered pests.
- The PPQ Cooperative Agricultural Pest Survey Program: Provides lists of National Pests of Concern and State Pests of Concern.
- A list of fungal plant pathogens for each U.S. state is currently being developed by PPQ to help expedite the permit process for obtaining research isolates. The list will be based on the Widely Prevalent Fungi of the United States Web site.
- Appendix B, Section B.4, of this manual also provides lists of bacterial, fungal, and viral plant pathogens that may be regulated by USDA.
- I.2.2.2 APHIS Animal Health Permits
- The Veterinary Services (VS) branch of APHIS protects and improves the health, quality, and marketability of our nation’s animals, animal products, and veterinary biologics by preventing, controlling, and/or eliminating animal diseases, and monitoring and promoting animal health and productivity. VS provides the following information on permits, types of materials, and diseases:
- VS animal health permits for importing controlled material, organisms, vectors, animal products, cell cultures and their products, live animals, semen, and embryos.
- Center for Import Export (NCIE) in APHIS VS regulates the import, export, and interstate movement of all animals and animal products (e.g., tissues, blood, and semen), including those that are genetically engineered.
- Center for Veterinary Biologics (CVB) in APHIS VS regulates and requires veterinary biologics permits for veterinary biologics. Examples of veterinary biologics include vaccines, antibodies, diagnostic kits, and certain immunomodulators, including those developed using genetically engineered organisms.
- Animal health – animal diseases information
- Animal diseases by animal species
- I.2.2.3 APHIS Genetically Engineered Organisms Permits
- APHIS uses the term biotechnology to mean the use of recombinant DNA technology, or genetic engineering (GE) to modify living organisms. APHIS regulates certain GE organisms that may pose a risk to plant or animal health. In addition, APHIS participates in programs that use biotechnology to identify and control plant and animal pests. Below is a list of the regulatory agency branches and requirements for genetically engineered organisms and facilities.
- Biotechnology Regulatory Services (BRS) in APHIS uses permits, notifications, and petitions to regulate the importation, interstate movement, or environmental release of certain GE organisms, including plants, insects, or microbes that may be plant pests. When transgenic Drosophila developed for research need to be moved, BRS requires a Drosophila Courtesy Permit Application or an APHIS 2000 Form to confirm they are not plant pests and therefore do not need to be regulated.
- See NCIE and CVB in Section I.2.2.2 above.
- I.2.3 CDC Agents or Vectors of Human Disease Permits
- CDC requires a U.S. Public Health Service permit to import an etiologic agent, or material containing an etiologic agent, host, or vector of human disease. A permit is also required for interstate transfer if the original CDC import permit was issued on the condition that any subsequent transfer of the material would require a permit. According to the CDC Etiologic Agent Import Permit Program, the materials listed below require a permit.
- Etiologic agents. Etiologic agents that are microorganisms, infectious agents, and toxins that cause disease in humans (e.g., bacteria, bacterial toxins, viruses, fungi, rickettsiae, protozoans, and parasites) require a CDC permit. Etiologic agents also include naturally occurring, bioengineered, or synthesized components of an etiologic agent when the component causes human disease. Examples of etiologic agents are listed in Appendix B, Section B.2 and Section B.3.
- Biological materials. Biological materials that are known or suspected of containing an etiologic agent also require a CDC permit. Examples include unsterilized specimens of human and animal matter (e.g., tissue, blood, body discharges, fluids, excretions or similar material) known or suspected of containing an etiologic agent.
- Hosts and vectors
- Animals. Any animal known or suspected of being infected with an organism capable of causing disease that is transmissible to humans may require a CDC permit. See the CDC Animal Importation Web site for more information.
- Bats. All live bats require an import permit from the CDC and the U.S. Fish and Wildlife Services.
- Arthropods. Any living insect or other arthropod known for, or suspected of, containing an etiologic agent requires a CDC permit.
- Snails. Snail species capable of transmitting a human pathogen require a CDC permit.
- I.2.4 Food and Drug Administration Import Program
- With the exception of most meat and poultry, all food, drugs, biologics, cosmetics, medical devices, and electronic products that emit radiation are subject to examination by the U.S. Food and Drug Administration (FDA) when they are being imported or offered for import into the U.S. Most meat and poultry products are regulated by USDA. FDA requires various notifications or approvals prior to importing. See the FDA Import Program Web site for more information.
- I.2.5 Fish and Wildlife Service Permits
- The import, export, or re-export of a wildlife or plant specimen may be regulated by a conservation law or treaty (e.g., Endangered Species Act) implemented by the U.S. Fish & Wildlife Service (FWS). These laws are part of domestic and international conservation efforts to protect wildlife and plants subject to international trade. Wildlife is any living or dead wild animal, its parts, and products made from the animal. Wildlife not only includes mammals, birds, reptiles, amphibians, and fish, but also invertebrates such as insects, crustaceans, arthropods, mollusks, and coelenterates. The FWS Permits Web site should be used to determine whether a wildlife or plant specimen requires a permit and how to obtain a permit. Table I-1 below provides examples of wildlife or plant specimens that may require a permit to export or import.
- Table I-1. Wildlife or Plant Specimens That May Require an FWS Permit
Export |
Import |
|
|
- Source: adapted from the UNH Shipment of Biological Materials Manual,
University of New Hampshire, March 30, 2007.
- I.3 Exporting or Transfer from the U.S.
- Controls for exporting from LBNL are outlined in the Berkeley Lab Export Control Manual. These export controls are designed to protect items and information that are important to the U.S. The controls are based on government rules and regulations that govern the transfer of the following items to non-U.S. entities or individuals, regardless of where or how the transfer takes place:
- Goods (systems, components, equipment, or materials)
- Technologies (technical data, information, or assistance)
- Software/codes (commercial or custom)
- The Berkeley Lab Export Control Manual should be consulted for general export control requirements. This section of the Biosafety Manual only outlines U.S.-based regulatory restrictions and lists related to the export of biological materials.
- Depending on the nature of the biological material, there may be restrictions or U.S. export permits required for the transfer of material to foreign countries. The country to which the material is being transferred may also require an import permit. If the material requires an export permit, the permit must be obtained from the appropriate government agency prior to transfer or shipment.
- When leaving the U.S., travelers may be questioned or packages may be opened and inspected by any inspection service provided by other countries. The person exporting the material should therefore:
- Obtain an export permit from the appropriate government organization prior to shipment, if required
- Package and label the material according to permit and shipping requirements
- Consider including a courtesy letter (e.g., a letter that describes the contents in detail and any hazards, concerns, permit requirements or lack thereof) with the shipment
- Several agencies and export control lists outlined in the next sections are involved in controlling exports of biological agents that may be used as biological weapons. Since LBNL is not a Department of Energy (DOE) Defense Programs laboratory, the export controls of most relevance at LBNL are those administered by the Department of Commerce, Bureau of Industry and Security, under the Commerce Control List (see Section I.3.1 of this program).
- I.3.1 Commerce Control List
- The Department of Commerce controls the export of all goods, technologies, and software not regulated by another government agency. Because LBNL is not a DOE Defense Programs laboratory, the most relevant export controls are those administered by the Department of Commerce – Bureau of Industry and Security (BIS), which maintains the Export Administration Regulations (EAR) Database. An important component of EAR is the Commerce Control List (CCL), a section of the regulations that lists specific goods, technologies, and software, the countries to which those items may or may not be exported, and any special restrictions or exceptions that may apply.
- A permit may be required from the Commerce Department when exporting biological agents such as human, animal, and plant pathogens or toxins; genetic elements and genetically modified organisms; and products that might be used for culturing large amounts of agents. See Table I-2 for an example list of biological agents on the CCL. Consult the most recent online list in CCL Supplement No. 1 to Part 774 Category 1. Consult the BIS CCL Web site and Berkeley Lab Export Control Manual for additional information.
Table I-2. Commerce Control List of Biological Agents
Human Pathogens and Toxins |
|
Bacteria
Viruses
Fungi
Toxins
|
Viruses (continued)
Toxins (continued)
|
- Table I-2. Commerce Control List of Biological Agents (Continued)
Animal Pathogens and Toxins |
|
Viruses
|
Viruses (continued)
|
- Table I-2. Commerce Control List of Biological Agents (Continued)
Plant Pathogens |
|
Bacteria
Viruses
|
Fungi
|
- Table I-2. Commerce Control List of Biological Agents* (Continued)
Genetic Elements and Genetically Modified Organisms |
|
Technical Notes:
|
Technical Notes (continued):
|
Note: Table is current as of July 2013. Source: adapted from CCL Supplement No. 1 to Part 774 Category 1, pages 58 to 63 (July 16, 2013); and UNH Shipment of Biological Materials Manual (March 30, 2007). |
- I.3.2 U.S. Munitions List
- It is unlikely that agents and substances on this munitions list would be used or exported from LBNL, but this section is provided so that personnel can understand what is covered by this list. The U.S. Department of State controls the export of “defense articles and defense services” under the International Traffic in Arms Regulations (ITAR). Items in this category to be export-controlled are placed on the U.S. Munitions List (USML), a section of ITAR (Part 121) maintained by the U.S. State Department in conjunction with the U.S. Department of Defense.
- The USML contains many categories of articles, including Category XIV (Toxicological Agents, Including Chemical Agents, Biological Agents, and Associated Equipment). Section (b) of this USML category states that biological materials include “Biological agents and biologically derived substances specifically developed, configured, adapted, or modified for the purpose of increasing their capability to produce casualties in humans or livestock, degrade equipment, or damage crops.” Such agents and substances are not typically used at LBNL, but the export of any item on the USML requires an export license issued by the U.S. State Department. Exports of all other products not covered by the USML are subject to the export jurisdiction of the U.S. Department of Commerce, BIS, as discussed in Section I.3.1 of this program.
- I.3.3 Biological Weapons Convention Lists
- The Convention on the Prohibition of the Development, Production, and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction, commonly known as the Biological Weapons Convention (BWC), has been in force since 1975. The BWC is the first multilateral disarmament treaty banning an entire category of weapons. It effectively prohibits the development, production, acquisition, transfer, retention, stockpiling, and use of biological and toxin weapons. The BWC is also a key element in the international community’s efforts to address the proliferation of weapons of mass destruction. The U.S. and other countries participating in the Australia Group (AG)are States Parties to the BWC. The AG is an informal forum of countries that, through the harmonization of export controls, seeks to ensure that exports do not contribute to the development of chemical or biological weapons.
- The AG maintains the following Common Control Lists of equipment and agents that require export control:
- Chemical weapons precursors
- Dual-use chemical manufacturing facilities and equipment and related technology and software
- Dual-use biological equipment and related technology and software
- Biological agents
- Plant pathogens
- Animal pathogens
- U.S. export permits or licenses are not directly regulated by the AG nor covered by the BWC lists, since the BWC lists are related to international treaty and are not derived from U.S. regulations. It appears to the author of this section of the ESH Manual Biosafety Program that the Department of Commerce BIS and U.S. Department of State are the U.S. agencies that have primary responsibility for enforcing U.S. exports related to the BWC. Section I.3.1 and Section I.3.2 above should therefore be used to determine U.S. regulatory requirements related to the BWC lists.
- The Core List of agents on the AG Common Control List appears to be the same or very similar to the agents on the BIS CCL presented above in Table I-2. Therefore, the Core List of agents on the BWC list is not relisted in this section of the Biosafety Program. However, the AG Common Control Lists also include a few additional agents that are not on the Core List. These additional agents are listed in Table I-3. It is not clear to the LBNL Biosafety Office, the author of this section, how or if these additional agents are regulated for U.S. export control.
- Table I-3. BWC Agents Not On the Commerce Control List
Plant Pathogens – Items for Inclusion in Awareness-Raising Guidelines |
||
Bacteria
Viruses
|
Fungi
|
|
Warning List 1 |
||
Bacteria
|
Fungi
|
- Note: Table is current as of July 2013. Source: The AG Common Control List of biological agents (March 2013), animal pathogens (July 2013), and plant pathogens (June 2012).
- Table Footnotes:
- Biological agents are controlled when they are an isolated live culture of a pathogen agent, a preparation of a toxin that has been isolated or extracted from any source, or material including living material that has been deliberately inoculated or contaminated with the agent. Isolated live cultures of a pathogen agent include live cultures in dormant form or in dried preparations, whether the agent is natural, enhanced, or modified. An agent is covered by this list except when it is in the form of a vaccine. A vaccine is a medicinal product in a pharmaceutical formulation licensed by, or having marketing or clinical trial authorization from, the regulatory authorities of either the country of manufacture or of use, which is intended to stimulate a protective immunological response in humans or animals in order to prevent disease in those to whom or to which it is administered.
- AG recognizes that this organism is ubiquitous. However, since it has been acquired in the past as part of biological warfare programs, it is worthy of special caution.
- It is the intent of Australia Group members to add to the control list strains of species of Clostridium identified as producing botulinum neurotoxin.
- I.4 References
- Berkeley Lab Export Control Manual
- Commerce Control List, Supplement No. 1 to Part 774 Category 1
- DOE Guidelines on Export Control and Nonproliferation, July 1999
- UNH Shipment of Biological Materials Manual, University of New Hampshire, updated March 30, 2007
- Web sites and Wikipedia articles of referenced government agencies and topics, accessed April 2010
- ____________________