TB Lab Guidelines | Table 1 & 2

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Goals for Working Safely with Mycobacterium tuberculosis
in Clinical, Public Health, and Research Laboratories

Table 1-Laboratory safety requirements
Table 2-Measures for controlling the risk for laboratory acquired tuberculosis
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TABLE 1. Laboratory safety requirements for persons who manipulate Mycobacterium tuberculosis complex species

ATS
Level BSL
Activity
Facility Safety equipment Practices and procedures

l 2

Collecting clinical specimens (including aerosol-induced sputa).

Transporting specimens to a higher level laboratory for isolation and identification.

Preparing and examining smears of killed tubercle bacilli for presumptive diagnosis and or following the progress of tuberculosis patients on chemotherapy.^*

BSL-2 and/or BSL-3 requirements including the availability of:

a hand washing sink.

an autoclave.

an eyewash facility

All specimens from patients suspected of having tuberculosis must be handled in a Class I or Class ll BSC.

Personal protective equipment must be used as indicated (see *Personal Protective Equipment).

Standard microbiological practices including:

Limited access to the laboratory

Biosafety manual available describing procedures for waste decontamination, emergency responses, and medical surveillance policies

Adherence to "sharps" precautions. ^#

Annual tuberculin skin test for all laboratorians.

ll 3 Performing functions of ATS Level l laboratory^§and

Processing specimens as necessary for microscopy and culture on standard egg- or agar-based media.

Identifying M. tuberculosis.

Performing optional drug susceptibility studies against M. tuberculosis.

Retaining mycobacterial cultures for additional or repeat tests (for up to 6 months).

BSL-3 facility requirements including:

Physical separation from access corridors

Access via two self-closing doors (e.g., through an anteroom, or a BSL-2 area).

Single-pass air system; exhaust air not recirculated.

Directional air flow through the laboratory following a negative pressure gradient.

Class ll or lll BSCs must be used for all manipulations of specimens and cultures that may contain M. tuberculosis. Personal protective equipment required includes gloves gown/lab coat, and respirator; eye protection required for persons who wear contact-lenses. BSL-3 practices and procedures including:

Controlled access to laboratory.

Decontamination of all waste before removal from the laboratory.

Personal protective equipment removed before leaving the laboratory.

Decontamination of laboratory clothing before laundering or disposal.

Baseline serum stored (for bloodborne-pathogen surveillance procedures).

lll 3 Performing functions of ATS level l and ll laboratories including:

Identifying all Mycobacterium species from clinical specimens

Performing required drug susceptibility studies against mycobacteria.

Conducting research and providing training to other laboratorians

Same as for ATS Level ll. Same as for ATS Level ll. Same as for ATS Level ll.

* Proficiency in reading smears may be maintained by examination of 10-15 specimens per week.
#These precautions include a) no recapping of needles, and b) use of puncture- and leak-proof waste containers.
§ Proficiency in culture and identification of M. tuberculosis may be maintained by digestion and culture of 20 specimens per week.
NOTE: ATS=American Thoracic Society; BSL=Biosafety Level; BSC=Biosafety Cabinet.

TABLE 2. Measures for controlling the risk for laboratory acquired tuberculosis

Activity Risk factors Administrative controls Practices and procedures BSL

Staining specimen spears for AFB without culture Centrifugation and manipulation of specimen may produce infectious aerosols. Kill tubercle bacilli. Treat specimen with equal volume of 5% hypochlorite solution; process in BSC; use aerosol- containing safety cups for centrifugation. 2

Preparing specimens for centrifugation and AFB culture Suspect specimens contain limited numbers of AFB and many are negative; set-up procedures involve potential for aerosolization. Train personnel in applicable safety procedures. Conduct all work in the BSC on a towel moistened with a tuberculocidal agent; use aerosol- containing safety cups for centrifugation. 2

Centrifugation of specimens suspected of containing live tubercle bacilli Centrifugation and manipulation of specimen may produce aerosols. Use biocontainment devices. Use aerosol-containing safety cups for centrifugation; open in BSC. 3

Inoculating cultures from specimens Production of aerosol during inoculation procedures. Use BSC and rigorously follow BL-3 practices and procedures. Follow aseptic techniques; autoclave all wastes from the BSC 2

Handling unopened primary- isolation plates or tubes Tubercle bacilli multiply with a generation time of 18-24 hours Treat all cultures as potentially infectious. Seal plates in gas-permeable bags. or with gas-permeable tape. Avoid aerosolization of inoculated liquid medium even if growth is not evident. 2

Staining smear of material from culture Many organisms; possible survival on slide, but low probability for aerosolization. Prepare slides in a BSC. Before removal from BSC, heat-fix (149-167° F
[65 C-75° C] for 2 hrs.) to kill tubercle bacilli. 3

Manipulating grown cultures of M. tuberculosis complex species on solid medium Colonies on solid medium contain greater numbers of bacilli than are present in sputum specimens, but aerosol potential is low. Vessels identified as containing M. tuberculosis complex; plates bagged or taped and screw-caps tightened. Use carts to safely transfer all cultures; open inoculated plates and tubes only in BSC. Use disposable loops; if not available, clean loops and needles in sand alcohol, then flame. 3

Transferring large volumes of cultures or suspensions of bacilli Substantial numbers of tubercle bacilli; high potential for aerosol generation when suspended in fluids, especially if clumps of bacilli are well dispersed; vortexing, sonicating, or vigorous mixing with a dispersant such as Tween 80 lead to aerosol production. Ensure BSC is certified annually using calibrated instruments by person certified by National Sanitation Foundation; maintain directional air flow and room air changes; develop spill protocol for management of accidents. Vortex and sonicate suspensions in BSC in closed tubes that are opened only in BSC. Use aerosol-containing centrifuge cups and open only in BSC. Manage waste safely. 3

Disposing of cultures of M. tuberculosis complex Handing material contaminated with tubercle bacilli outside BSC by untrained persons Identify material with proper disposal labels and autoclave prior to disposal. Discard liquid waste into a tuberculocidal disinfectant solution; noncompressible discard containers used in BSC should contain 2-3 cm of tuberculocidal disinfectant inside a plastic liner which is covered before transfer to autoclave. 3

Conducting research on M. tuberculosis complex species May employ large volumes of fluids containing high concentration of bacilli and high-risk aerosolizing procedures. Ensure compliance with all biosafety recommendations Maintain all elements of BSL-3 3

Shipping cultures or specimens of M. tuberculosis complex Potential exposure if package leaks or breaks. Provide shipping containers approved by Department of Transportation Ship in triple - packaged container. Follow PHS regulations for transport of diagnostic specimens and infectious substances.

Studying animals infected with M. tuberculosis complex species Aerosols may be created during inoculation; bedding may contain viable bacilli in dried urine and feces; generation of droplet nuclei by coughing of nonhuman primates. Provide containment cages; use proper facilities (see text). Use bonnet-top rodent cages at ABSL-2; change cages in BSC. Houses nonhuman primates at ABSL-3 and wear respirators when in room. 2 and/or 3

Note: BSL=Biosafety Level; AFB=Acid-fast bacilli; BSC=Biological Safety Cabinet; NA=Not Applicable; ABSL=Animal Biosafety Level.

Office of Health and Safety, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Mail Stop F05 Atlanta, Georgia 30333, USA Last Modified: 1/2/97
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ATS Level	BSL	Activity	Facility	Safety equipment	Practices and procedures
l	2	Collecting clinical specimens (including aerosol-induced sputa). Transporting specimens to a higher level laboratory for isolation and identification. Preparing and examining smears of killed tubercle bacilli for presumptive diagnosis and or following the progress of tuberculosis patients on chemotherapy.^*	BSL-2 and/or BSL-3 requirements including the availability of: a hand washing sink. an autoclave. an eyewash facility	All specimens from patients suspected of having tuberculosis must be handled in a Class I or Class ll BSC. Personal protective equipment must be used as indicated (see *Personal Protective Equipment).	Standard microbiological practices including: Limited access to the laboratory Biosafety manual available describing procedures for waste decontamination, emergency responses, and medical surveillance policies Adherence to "sharps" precautions. ^# Annual tuberculin skin test for all laboratorians.
ll	3	Performing functions of ATS Level l laboratory^§and Processing specimens as necessary for microscopy and culture on standard egg- or agar-based media. Identifying M. tuberculosis. Performing optional drug susceptibility studies against M. tuberculosis. Retaining mycobacterial cultures for additional or repeat tests (for up to 6 months).	BSL-3 facility requirements including: Physical separation from access corridors Access via two self-closing doors (e.g., through an anteroom, or a BSL-2 area). Single-pass air system; exhaust air not recirculated. Directional air flow through the laboratory following a negative pressure gradient.	Class ll or lll BSCs must be used for all manipulations of specimens and cultures that may contain M. tuberculosis. Personal protective equipment required includes gloves gown/lab coat, and respirator; eye protection required for persons who wear contact-lenses.	BSL-3 practices and procedures including: Controlled access to laboratory. Decontamination of all waste before removal from the laboratory. Personal protective equipment removed before leaving the laboratory. Decontamination of laboratory clothing before laundering or disposal. Baseline serum stored (for bloodborne-pathogen surveillance procedures).
lll	3	Performing functions of ATS level l and ll laboratories including: Identifying all Mycobacterium species from clinical specimens Performing required drug susceptibility studies against mycobacteria. Conducting research and providing training to other laboratorians	Same as for ATS Level ll.	Same as for ATS Level ll.	Same as for ATS Level ll.

Activity	Risk factors	Administrative controls	Practices and procedures	BSL
Staining specimen spears for AFB without culture	Centrifugation and manipulation of specimen may produce infectious aerosols.	Kill tubercle bacilli.	Treat specimen with equal volume of 5% hypochlorite solution; process in BSC; use aerosol- containing safety cups for centrifugation.	2
Preparing specimens for centrifugation and AFB culture	Suspect specimens contain limited numbers of AFB and many are negative; set-up procedures involve potential for aerosolization.	Train personnel in applicable safety procedures.	Conduct all work in the BSC on a towel moistened with a tuberculocidal agent; use aerosol- containing safety cups for centrifugation.	2
Centrifugation of specimens suspected of containing live tubercle bacilli	Centrifugation and manipulation of specimen may produce aerosols.	Use biocontainment devices.	Use aerosol-containing safety cups for centrifugation; open in BSC.	3
Inoculating cultures from specimens	Production of aerosol during inoculation procedures.	Use BSC and rigorously follow BL-3 practices and procedures.	Follow aseptic techniques; autoclave all wastes from the BSC	2
Handling unopened primary- isolation plates or tubes	Tubercle bacilli multiply with a generation time of 18-24 hours	Treat all cultures as potentially infectious.	Seal plates in gas-permeable bags. or with gas-permeable tape. Avoid aerosolization of inoculated liquid medium even if growth is not evident.	2
Staining smear of material from culture	Many organisms; possible survival on slide, but low probability for aerosolization.	Prepare slides in a BSC.	Before removal from BSC, heat-fix (149-167° F [65 C-75° C] for 2 hrs.) to kill tubercle bacilli.	3
Manipulating grown cultures of M. tuberculosis complex species on solid medium	Colonies on solid medium contain greater numbers of bacilli than are present in sputum specimens, but aerosol potential is low.	Vessels identified as containing M. tuberculosis complex; plates bagged or taped and screw-caps tightened.	Use carts to safely transfer all cultures; open inoculated plates and tubes only in BSC. Use disposable loops; if not available, clean loops and needles in sand alcohol, then flame.	3
Transferring large volumes of cultures or suspensions of bacilli	Substantial numbers of tubercle bacilli; high potential for aerosol generation when suspended in fluids, especially if clumps of bacilli are well dispersed; vortexing, sonicating, or vigorous mixing with a dispersant such as Tween 80 lead to aerosol production.	Ensure BSC is certified annually using calibrated instruments by person certified by National Sanitation Foundation; maintain directional air flow and room air changes; develop spill protocol for management of accidents.	Vortex and sonicate suspensions in BSC in closed tubes that are opened only in BSC. Use aerosol-containing centrifuge cups and open only in BSC. Manage waste safely.	3
Disposing of cultures of M. tuberculosis complex	Handing material contaminated with tubercle bacilli outside BSC by untrained persons	Identify material with proper disposal labels and autoclave prior to disposal.	Discard liquid waste into a tuberculocidal disinfectant solution; noncompressible discard containers used in BSC should contain 2-3 cm of tuberculocidal disinfectant inside a plastic liner which is covered before transfer to autoclave.	3
Conducting research on M. tuberculosis complex species	May employ large volumes of fluids containing high concentration of bacilli and high-risk aerosolizing procedures.	Ensure compliance with all biosafety recommendations	Maintain all elements of BSL-3	3
Shipping cultures or specimens of M. tuberculosis complex	Potential exposure if package leaks or breaks.	Provide shipping containers approved by Department of Transportation	Ship in triple - packaged container. Follow PHS regulations for transport of diagnostic specimens and infectious substances.
Studying animals infected with M. tuberculosis complex species	Aerosols may be created during inoculation; bedding may contain viable bacilli in dried urine and feces; generation of droplet nuclei by coughing of nonhuman primates.	Provide containment cages; use proper facilities (see text).	Use bonnet-top rodent cages at ABSL-2; change cages in BSC. Houses nonhuman primates at ABSL-3 and wear respirators when in room.	2 and/or 3