| FACT
SHEET
Biosafety Level 2+ (Lentiviral/Retroviral Vectors)
CONTAINMENT
LEVEL:
• Consistent Biosafety Level 2 practices are adopted and rigorously
followed. Some BL3 practices are also adopted. |
FACILITY:
• The Principal Investigator must designate a laboratory that
contains a biosafety cabinet, incubator, centrifuge, microscope, CO2
source. It is preferable that this be an inner lab with 2 doors between
the BSC and the hallway. Restricted entry to the lab. Air must flow
from the hallway to this lab (negative to the hallway) and all air
is exhausted outside the building, not recirculated. |
SIGNAGE:
• A sign with the following information is posted in these areas:
Staff should be aware that there is potential for recombinant virus
generation. All experimental materials must be handled with great
care. Sharps must be eliminated from experimental procedures; in particular,
procedures involved in pelleting virus should be reviewed carefully. |
PPE:
• Gloves and lab coat; gloves must be pulled over the knit cuffs
of a disposable lab gown which ties in the back and is liquid resistant.
Double gloving should be considered, depending upon the procedures
being performed. Standard laboratory safety dress code should be followed
(no shorts or open toed shoes, etc.). |
WORK
PRACTICES:
- Close laboratory
doors
- Use certified
biological safety cabinet for all manipulations
- Load and
unload ultracentrifuge buckets in biosafety cabinet; screw on
covers, ensuring that O-ring is in place and flexible, spray outside
with approved quaternary ammonium disinfectant.
- Do not use
"sharps" to harvest virus pellet. No "sharps"
(needles, glass Pasteur pipets) may be used with these cultures.
Plastic aspiration pipets are a replacement for glass Pasteur
pipets.
- For Aspiration-
Use plastic vacuum flask with a second vacuum flask connected
to it as a backup, with non-collapsible tubing capable of withstanding
disinfection. To the second vacuum flask attach a hydrophobic
and a HEPA filter (or combination filter) so that nothing is sucked
into the house vacuum system. These 3 items are attached in series
from the vacuum source in the hood or a vacuum pump (Example)
- Cells must
be placed in a dedicated incubator, labeled "lentivirus
(or retrovirus) experiments ONLY"
- Cells exposed
to lentiviral (or retroviral) vectors are not removed from (lab
room # ) for experimental purposes unless inactivated by approved
procedures.
- Undiluted
bleach should be pulled into the aspirator flask using suction
to decontaminate tubing. Final concentration in the aspirator
flask when it is 2/3 full should be at least 10% bleach/0.5% NaOCl.
- Within the
biological safety cabinet (BSC):
Dispose
of plastic pipet tips and serological pipettes in a cardboard
biohazard box. This box is put into an autoclave bag with any
other biohazardous waste in the BSC, closed, the outside of
the autoclave bag is sprayed with a disinfectant approved for
efficacy against lentivirus, then removed from the BSC, put
into another, clean autoclave bag, and subsequently autoclaved
at conditions appropriate for decontamination (see UK autoclave
procedures)
- Biosafety
cabinet surfaces must be thoroughly wiped with disinfectant approved
for efficacy against lentivirus or retrovirus.
- Biohazard
waste containers should be hard sided, with a foot operated lid,
and easily decontaminated with liquid disinfectant. The autoclave
bag should be folded over the rim so that only the inside of the
bag is visible.
- All procedures
should be carried out according to the guidelines outlined by
the CDC and NIH for work under BSL2 containment with BSL3 procedures.
|
References:
1. Donald E. Mosier,
Ph.D., M.D , The Scripps Research Institute, “Safety Considerations
for Retroviral Vectors: A Short Review”
2. Applied Biosafety, vol. 7, 2002 |
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