This document summarizes information found in our Institutional Biosafety Committee Protocol and Animal Biosafety Protocol. The official IBC protocol and/or any PSU-specific safety guidance supersedes what is written here and this document may be out of sync with the official IBC protocol.
We have a mixture of BSL1 and BSL2 micro-organisms in the lab. You can tell the biosafety level of any strain you may be using by viewing the lab strain collection spreadsheet available via Slack. BSL2 organisms are known to be pathogens and pose a moderate-risk to users.
Human feces, even when collected from healthy donors, may harbor a variety of pathogens of bacterial, viral, and parasite origin. Feces should be treated with universal precaution as we can't be 100% sure of what it could contain.
Mice which have been colonized with BSL2 agents pose a special risk. They must be adequately contained and only handled within a biosafety cabinet. Feces from ABSL2 animals should be handled as we handle human feces.
Human cell lines may also be a biological safety concern and should be handled as BSL2.
Recombinant DNA and organisms carrying it need to be treated as BSL2 agents to prevent their spread into the environment. Especially when that recombinant DNA encodes bacterial toxins or components thereof.
We have 3 types of hoods in the lab: biosafety cabinets, chemical fume hoods, and PCR laminar flow hoods. Neither the chemical fume hood or PCR hood are intended to control biological hazards, and in the case of the PCR hood, offer the user no protection. Air going in and out of the BSC is filtered to remove contaminants and protect both you, and the sample you are working with. Air from the chamber is exhausted out of the building.
To use the BSC, turn it on 5-10 minutes ahead of time and turn on the UV light (do not look directly at the UV light). Make sure the back of the BSC is clear to allow proper air flow. Avoid sudden motions in and out of the BSC which could disturb airflow and avoid walking directly behind someone using the BSC which may disrupt air flow. When you are done with the BSC, spray and wipe surfaced with 70% ethanol, and turn on the UV light for >=15 minutes.
Note: PSU does not recommend the use of UV as a primary method of disinfection and UV light can be blocked simply by plastic (like petri dish lids). Think of it more as an extra layer of defense with the wiping of the hood with ethanol as the primary method of cleaning.
See PSU policity SY29.
Liquid waste (examples: culture supernatants, blood, fecal extracts) can be disposed of in two ways: inactivate with sodium hypochlorite (bleach) or by autoclave:
Option 1: Autoclave. Collect liquid waste in autoclavable container which is no more than half full (for example a pyrex bottle). Autoclave on a liquid cycle which has a sterilization time of at least 30 minutes. After the waste has cooled, it can be disposed of down the sink. Note: do not add bleach if you will autoclave as autoclaving bleach-containing waste can release toxic gasses. This is the preferred method for disposing of C. difficile liquid cultures.
Option 2: Bleach. Collect liquid waste and add 1/10th volume of bleach. Leave to disinfect for 1 hour and then pour down drain. Ensure bleach is <6 months old and has a concentration of at least 5.25% sodium hypochlorite (Note: Clorox is 8.25%). If the culture contains purified spores from C. difficile either autoclave instead or add 2% NaOH to bleach (can use sodium hydroxide pellets). Do not leave bleached cultures in the sink for more than 24h.
All waste which has come into contact with BSL2 agents (ex, petri dishes, pipette tips, gloves, etc) should be disposed of in the red biohazardous waste containers around the lab. When these are ~2/3 full, move them to the main biohazard waste pale in the lab. When the main waste is ~1/2 full, it should be sterilized in the autoclave using the 60 minute waste cycle on the autoclave. After autoclaving, it can be transferred to the white bins next to the autoclave. Never over-fill the waste as it makes it dangerous to handle for disposal!.96 well plates used for MIC assays can be treated as solid waste.
All sharps (ex. needles or razor blades) should be disposed of in hard-shelled sharp containers around the lab. Never fill these containers above the fill line!
Animal carcasses must be double bagged with the outermost bag being a red autoclavable bag identifying the contents as biohazardous. These may be temporarily stored in lab -20˚C freezers until being returned to South Frear to the designated freezer for incineration.
Lab coats are available for everyone in the lab. If these become soiled for any reason, place into the lab coat collection container and take a new lab coat.
The lab has a supply of nitrile gloves in all sizes. Nitrile is the preferred material for most applications; however, if you require a different material or glove style, please bring this to my attention!
There is a supply of safety glasses above the hand washing sink to which you can help yourself.
We do not currently have respirators available in the lab as we are not working with any agents which would require them. Conduct all work that could generate aerosols in the BSC or anaerobic chamber.
All manipulations of BSL2 aerobes which could create aerosols (ex vortexing) should be conducted in a Biosafety Cabinet to prevent spread. When BSL2 agents are centrifuged, be sure to use a centrifuge which has the appropriate aerosol-containing rotors/safety cup such as any of our microcentrifuges. The user should wear appropriate PPE (nitrile gloves, lab coat and glasses) and avoid blocking the entire back of the hood which can disrupt air flow. Discard all materials inside the BSC into the red biohazardous disposal bin inside the hood. When you finish your work, wipe down the work surfaces with 70% Ethanol and turn the UV light on for at least 30 minutes. Discard gloves in biohazardous trash. Cultures can be grown in the incubators in 304C; however, manipulations of said cultures should be always done in the biological safety cabinet.
Manipulation of facultative anaerobes or aerotolerant anaerobes should be conducted as per Aerobes in a biological safety cabinet. For obligate anaerobes, manipulate all cultures inside of Coy Anaerobic Chamber. Before and after work, wipe all surfaces down with 100% isopropanol. All materials which contact cultures inside the coy should be discarded into the biohazardous bin inside the anaerobic chamber. Liquid cultures in the anaerobic chambers should be bleached before they are taken out of the exchange gasket. Empty the anaerobic chamber's biohazardous waste into the main lab's biohazardous waste for disposal above.
Animals will be housed within isolators which will serve as containment for inoculated animals. Materials such as soiled bedding, leftover feed, water, and caging will be autoclaved upon removal from the isolator. When animals or samples are removed from the isolator, they will be placed in a sealed container which is decontaminated with chlorine dioxide (Exspore) during removal. Subsequent handling of animals and samples that are permanently removed from the isolator will be conducted within a biosafety cabinet. While working with animals, wear gloves, safety glasses, and a lab coat/gown.
When transporting ABSL2 animals, they must be transported in either cages with filter lids which are secured (example with tape) or in temporary transport containers which are secured in the event they tip over. Animals must be transported between buildings inside an insulated secondary container to prevent exposure to temperature extremes and to protect against animal escape. Both cages and/or transport containers must be appropriately labelled as biohazards.
If moving other BSL2 materials anywhere outside the lab or between buildings, they need to be in a secondary unbreakable container (for example a nalgene or tuperware) which contains sufficient absorbent materials to contain a spill. The secondary container needs to be labeled with a biohazard label.
- Wash your hands any time when you leave the lab.
- Wear appropriate clothing (no shorts and flip flops... this hurts me more than you I promise).
- Keep hair tied back to prevent it from catching on fire from bunsen burners.
- Do not flame solutions prepared in >50% ethanol as these will catch on fire (for example vitamin K stock). I have seen multiple people light their bench on fire doing this at other institutions. If you accidentally do this, move away from your bench and extinguish any flame on your person. Remove any flammable objects from near the ethanol fire. Assuming the volume is relatively small, the fire will burn out in a matter of seconds, otherwise evacuate the area and pull the fire alarm.
- Wash your hands before touching your computer (and/or clean your computer keyboard frequently with isopropanol).
- Don't touch your cell phone with your gloved hands
- Don't touch your cell phone with your gloved hands
- Don't touch your cell phone with your gloved hands
- Don't touch your cell phone with your gloved hands
- Don't touch your cell phone with your gloved hands
- Consider cleaning your cell phone on a regular basis using isopropanol
- Do not eat or drink anywhere inside the lab. You can transfer closed materials through the lab to the lunch room by my office for your enjoyment.
- Do not bleach nucleic acid extraction waste as it frequently contains guadinine isothiocynate which reacts with bleach to release cyanide gas. The act of nucleic acid extract is generally sufficient to kill anything in the sample and the waste from DNA extraction should be disposed of as chemical waste.
- Using old bleach solutions. Diluted bleach is not stable and can rapidly loose activity. Always use fresh bleach from the original container.