Biosafety Level 3 Laboratory (BL3)
The BL3 facility should be used routinely within working hours (8.00 – 17.00).
No unauthorized person should be permitted in the BL3 area. Certain knowledgeable visitors may be permitted to enter the BL-3 if accompanied by an authorized senior staff individual.
Personnel with prior HIV or BL-3 protocol experience will be cleared to work in the facility by a senior staff individual, following an observational period. The length of the training period will depend on the extent of previous experience and familiarity with all relevant techniques (such as tissue culture) and shall be determined by the designated senior staff individual.
All personnel should read relevant protocols and familiarize themselves with the standard protocols before beginning work in the BL3.
All laboratory personnel will be given the opportunity of an HIV test at the beginning of the work period and thereafter at intermittent intervals. Serum will be taken at the beginning for reference and baseline for HIV RNA levels. Personnel should be immunocompetent and fully covered for HBV and tetanus. Personnel will be given the option to meet with the medical officer in charge before commencing work in the BL3 facility, and at any time thereafter, to discuss any concerns and testing options available.
It is desirable that two individuals should be working in the BL3 when tissue culture is being performed. Should this not be possible, still it is mandatory that al least another person is present in the laboratory immediately outside of the BL3 area. This person will be responsible to monitor constantly the activity in the BL3 through the internal video equipment. No individual should feel obliged to work alone in the BL3.
A person with open wounds, which cannot be adequately covered, may not work in the BL3. Persons with a serious medical condition must discuss the situation with the medical officer in charge before entering the BL3 area.
Entering and leaving
Sandals, open-toed shoes and short trousers/skirts are not permitted in the BL3 area. All exposed skin must be covered with protective clothing. In the dressing-room of the BL3 appropriate protective clothing should be worn, these consist of a long lab coat, a disposable surgical gown, one pair of latex gloves, mask and shoe covers. Alternatively it is possible to wear disposable goggles and a surgical disposable mask. It is suggested to keep a pair of comfortable shoes in the BL3 (disposable shoe covers do not fit heavy winter shoes).
When leaving the BL3 module disposable gloves, shoe covers and gowns must be removed and disposed of in the contaminated waste. Goggles and masks should be kept in the BL3 and changed at intervals.
Use of the Biosafety cabinets
Check the biosafety cabinet airflow (red indicator light above the cabinet). The biosafety cabinets should be switch on by the first user and switch off by the last to leave the BL3 area. In case of power failure, the emergency generator will start.
The first person using the biosafety cabinet empties the bleach waste container for liquid waste and the biosafety container for solid waste: spray container with 70% ethanol before removing from cabinet. Put the solid waste in plastic bags and liquid in the container). After refilling the waste container with fresh bleach clean the surface of the hood with bleach, then rinse with water and then clean with 70% ethanol. This operation should be performed also at the end of the work, it is important to rinse with water because bleach is corrosive and will irreversibly affect the hood. Alternative detergents provided in th BL3 may be used instead.
Recommended contents in a hood:
• Sterile PBS
• Pipette aids
• Waste container for tips and pipettes
• Waste container for liquid waste (with bleach)
• Tray of blue tips.
• Tray of yellow tips.
• Eppendorf tubes
• Marker pen
• Pipettes (1 set)
When using the containment hoods a second pair of latex gloves should be worn and these should be removed before removing hands from the hood. Only one pair of gloves should be worn outside the hoods.
The metal grids should not be blocked so that the air curtain is properly maintained. No work in open vessels is to be conducted on the benchtop. All procedures are to be performed carefully, in order to minimize the creation of aerosols. Pelleted cells and viruses must be re-suspended by gentle pipetting in order to avoid the creation of aerosols. Pipetting must be performed slowly with the pipette kept below the surface of the liquid.
Pipetting devices must be used. Mouth pipetting is forbidden. The amount of materials stored in the module should be minimal. All buffers, media and other solutions should be prepared in the lab and brought in the BL3 module as needed. The hoods should not be cluttered with unnecessary objects.
Sharp items such as needles, Pasteur pipettes, pointed end scissors and razor blades are prohibited except where their need is essential ( e.g. tissue preparation). Whenever possible, use plasticware instead of glassware. All sharp objects that have to be used must be disposed of in the correct yellow disposal container.
Individuals should regard all items and reagents within the hoods as potentially biohazardous and take all the necessary precautions. Used pipettes and pipette tips are filled with ethanol or an alternative disinfectant such as bleach. Infected tubes, flasks or supplies should be sprayed with 70% ethanol prior to disposal. Closed containers can be disposed of in small plastic bags before autoclaving. Liquid waste should be disposed of in a sealed container with disinfectant.
Hoods, countertops and equipment should periodically be wiped down with alcohol (70%) after use. The interior of the hood should be kept clean at all times. All materials that come in contact with infectious substances must be decontaminated overnight in a container filled with bleach, these buckets are emptied in the morning and filled with new disinfectant. After finishing work in the hood, remove waste and put it in the plastic waste bag after spraying with ethanol.
Working with radioactive material in the BL3
Before commencing with any radioactive work, the project must first be discussed with the biosafety officer in charge to ensure that the radioactivity license is active. Inform also the rest of the personnel that you are working with radioactive material. Normal safety rules concerning working with radioactive materials should be applied.
Transport and inactivation of virions
• Removal of viable tissue culture materials, viral stocks etc is prohibited. Ask specific permission for special reasons.
• Removal of samples for further processing outside the BL3 facility requires an inactivation step to be carried out in the BL3 facility. Examples of common practices are given below. Any procedure other than those described below must be previously approved.
• Vials, ice buckets etc must be carefully wiped with 70% ethanol before exiting the BL3 facility.
• Any material originating from the BL3 and processed outside must be handled at BL2 with special care. Waste should be discarded in bags to be autoclaved.
Examples of protocols that inactivate HIV-1:
Procedures that inactivate retroviruses include fixation in formaldehyde, glutaraldehyde or acid-ethanol. Also, addition of NP-40 at >0.5% has been shown to render HIV undetectable after one minute (Resnick et al 1986). Heat inactivation remains the best method (56 °C for 30 minutes, 60 °C for 20 minutes). Guanidium treatment completely denatures proteins and inactivate virus.
Specific protocols are described briefly:
CAT assay (ref. Sodroski DFCI)
• harvest cells and wash 1X PBS
• resuspend cells cell pellet in 0.25 M Tris pH 8
• rapid freeze-thaw/vortex
• HEAT TO 60 °C FOR 20 MINUTES (heat treatment does not affect CAT assay, in many cell types treatment actually improves the assay)
• Remove samples from BL3 and process as usual
(ref. See CDC/OHS: inactivation of HIV at http://www.cdc.gov/od/ohs/biosfty/inacthiv.htm)
Formaldehyde concentration of 0.5% to 2% are effective in inactivation of HIV. A 30 minutes fixation in a buffered phormaldehyde solution before acquisition on the flow cytometer is typical.
Always use FRESH fixation solutions.
• harvest cells and wash 2X PBS
• resuspend cells in 2% buffered formaldehyde and leave under the hood at room temperature for 20 minutes
• wash 1X PBS an proceed with staining outside BL3
For immunoflorescence DO NOT introduce glass slides/coverslips in the BL3, these are sharp objects. Ask permission if you intend to do this kind of procedures.
The first step of the NEN ELISA procedure for HIV p24 requires treatment with a detergent (Triton X-100, 0.5% final) that inactivates HIV. However, all the ELISA procedure and incubations should be carried out in the BL3 up to the developing of the color (see detailed protocol found in the BL3 lab). Optical reading can be done out of the BL3.
DNA extraction (DNAeasy Tissue Qiagen )
• Harvest cells and wash 1XPBS
• Resuspend cells in 200 ul PBS
• Add RNase A, add proteinase K and buffer AL
• Mix, vortex, incubate at 70 °C for 10 minutes. THIS STEP INACTIVATES VIRUS
• Move to BL2.
RNA extraction (Rneasy Qiagen).
• Harvest cells and wash 1XPBS
• Resuspend pellet in RTL buffer (highly denaturing guanidine isothiocyanate buffer + b-mercaptoethanol). Be sure to resuspend the pellet by pipetting/vortexing several times. THIS STEP INACTIVATES HIV.
• Proceed with the RNA extraction in BL2. However, DO NOT USE NEEDLES to homogenize the sample and reduce viscosity. Use instead the Qiashredder spin columns.
for Western blots, lyse cells in Laemli buffer, heat to 95 °C 5 minutes, then proceed at BL2.
For IP make sure the lysis buffer contains NP40 > 0.5% to inactivate virus.
Use of plasmids containing HIV sequences
Full-length purified HIV proviral DNA is infectious. Infection could occur if such DNA is injected, or if it makes contact with broken skin or mucous membranes (mouth, eyes).
It is not allowed to work with HIV-1 molecular clones outside the BL3 lab. The only exception is in case the plasmid is used as a template in a PCR reaction. The PCR reaction should be prepared in the BL3 lab.
HIV vectors carrying the HIV env should be used in the BL3 and treated as wt HIV. Use of HIV vectors pseudotyped with VSV-G may be allowed at BL2 upon careful check of the characteristics of each vector variant (i.e. which accessory genes have been deleted, frequency of recombination etc.) and specific permission.
PROTOCOL VIOLATIONS SHOULD BE REPORTED TO THE BIOSAFETY OFFICER
Any equipment or instrument leaving the BL3 area must be wiped down 2x with 70% alcohol.
Centrifuges – spinning of tubes containing HIV should be performed in aerosol containers. Spinning of 24 or 96 well plates should only be done after correct balancing of the plates. The centrifuge should be periodically cleaned with 70% ethanol. After centrifugation, the buckets should be opened in one of the containment hoods. When using the ultracentrifuge the rotor should be opened inside a hood and the inserts CAREFULLY removed. The buckets and rotor should be extensively cleaned with 70% ethanol afterwards.
All non-radioactive waste must be autoclaved within 24hrs (program 03). Radioactive waste must be chemically disinfected (with alcohol or bleach) prior to disposal in the proper radioactive waste containers.
Solid waste is autoclaved in sealed plastic bags. Liquid waste containing bleach is maintained in a sealed container and disposed at intervals (call the biosafety officer). No bleach-containing material should be autoclaved to avoid corrosion of the autoclave. Chemical and organic waste should not be autoclaved to avoid hazardous fumes and should be disposed after bleach treatment (call the biosafety officer).
Cleaning and maintenance
Every week somebody is assigned to perform the so-called autoclave duty. The person having the autoclave duty is responsible for the opening and closing of the lab and disposal of the garbage (see below). Everybody, however, is responsible for cleaning and emptying of his/her working area after finishing with the experiments. It is also everybody’s responsibility to replace full waste bags and to refill boxes with pipette tips and Eppendorf tubes.
Replace stocks when using the last item, order if necessary. Replace stock solutions in time. Clean spills as soon as possible. Clean and empty the work area after you finished your experiments. Leave enough space in the cabinets if you have long incubation/centrifugation times so other people can work in between.
It is suggested that each week who is in charge of the autoclave duty should also be in charge of emptying of garbage cans and cleaning of the floor, lab-benches, tables, waterbaths, incubators, tabletop centrifuges and biosafety cabinets.
The air filters in the biosafety cabinets should be replaced at least once a year. This replacement is done early in the morning to make sure that at least 12 h has passed since the last use of the cabinet. The filters are autoclaved and disposed.
At the start of the day
– Replace waste containers in biosafety cabinets
– Seal waste bags and put them into the autoclave. Switch on autoclave and wait until pressure reaches 3-4. Check that the sensors inside the autoclave are immerged in water, if not refill the container.
– Perform autoclave preheating program (number 05)
– Autoclave plastic waste bags, filter tips and gowns if necessary (program number 03)
– Check and if necessary order liquid Nitrogen:
– Refill bleach tank(s). There should always be enough bleach present to refill the containers in the biosafety cabinets.
– Check water levels in incubators. Sterile, distilled water should be added to the waterbaths when necessary.
At end of the day
– Cabinets: put pipette aid on charger
Turn off lights in safety cabinets
Turn off vortex
– Turn off microscope(s) and place cover
– Turn off centrifuges, lights, etc
If the person doing the autoclave duty is not the last one leaving the lab, (s)he should shift the closing of the lab to the person still working. If unsure whether or not somebody is still working, close the lab anyway so that when somebody comes in later (s)he knows that (s)he should close the lab again.
Major spills – Major spills should not be handled by the individual who caused the spill. Individual must report the spill to who is present in the room in order to concentrate on its own decontamination. The contaminated surface should be extensively treated with bleach for at least 10 minutes using soaked paper or material. Then the surface should be rinsed with water and then treated with 70% ethanol. Paper, material and remains of the damaged culture should be put in a “biosafety” container and autoclaved or treated with bleach for at least 24 hours.
Minor spills – Handling of the minor spill should be the responsibility of the individual who caused the spill. The contaminated surface should be extensively treated with bleach. The contaminated materials should then be disposed of in biohazard bags, and the involved surface again treated as above. If clothes are contaminated then spray them with 70% ethanol before removing and then autoclave and discard.
In case of an accidental exposure to HIV , the affected mucosa or skin should be rinsed immediately and thoroughly with running water (sink, shower or eyewash). The affected skin area should also be rinsed with 70% alcohol or washed with soap and water. Do not traumatize the affected area any further. Promptly notify and seek the assistance of the medical officer in charge who can be reached at the telephone numbers listed below 24hrs a day. All accidents should be reported to the biosafety officer as soon as possible after treatment.
Formation of potentially hazardous aereosol (other than in fume hoods) . All personnel should leave the BL3 immediately. Access to the facility should be prohibited for at least one hour to allow the change of the air in the laboratory. Inform the biosafety officer in charge immediately. All personnel involved in the accident should be visited by the medical officer in charge.
Spills from rotor buckets . If you suspect problems during the run switch off the centrifuge and leave closed for 30′ to avoid exposure to aereosol. If the spill is discovered at the end of the run, close the cover and leave closed for 30′. Inform the biosafety officer in charge. For all subsequent operations personnel should wear robust gloves and use forceps with cotton wool to remove contaminated fragments from the instrument. Broken tubes, buckets and accessories should be treated with a non corrosive disinfectant for at least 24 hours and then autoclaved. Undamaged tubes should be treated with disinfectant for at least 60 minutes before opening.
Doors are blocked . Use the telephone to inform the biosafety officer in charge.
Electric blackout. The fan of the laboratory remains active to allow the mantainance of a negative pressure. After 20” the generator starts functioning. If autoclave is not functioning inform the biosafety officer in charge to restart.
Fire. Leave the lab and inform the biosafety officer immediately.
EMERGENCY CONTACT INFORMATION (Phone numbers etc) ARE PROVIDED IN THE BL3 FACILITY
For further information on this Facility contact:
Alessandro Marcello, PhD, Group Leader, Molecular Virology, ICGEB Trieste, Padriciano 99, 34149 Trieste, Italy
Tel: +39-040-3757384, fax: +39-040-226555, e-mail: