Virus lab, Biosafety level 2 Core facility at BioClinicum

• Only personnel that have retrieved the appropriate training can work in the room.
• Before you start working in the room make sure you are aware of current guidelines (should be dated).
• Each new project needs to have an approved application for “Anmälan av ny GMM-användning” (Ask Paula Mannström about how to apply for this).
• Using sharp objects while working in the room including glass pipettes, syringes and scalpels is not allowed. Plastic pipettes are ok.
• All large-scale spills and accidents must be reported to virus lab responsible personnel.
• There is a logbook by laminar hood in the lab that must be signed when working in the room with virus.

Access to the GMM-L room

Work with microorganisms is recognized by the use of specific labels "Biosafety Level 2" ("skyddsnivå 2") at the entrance. Biosafety Level 2 (BSL2) is appropriate when work is performed with most microorganisms that can cause disease in humans. It is the lowest biosafety level for work involving human pathogens. Only certified personnel can work in the laboratory. Working in the GMM-L room is restricted to persons who have received personal instructions from the virus room contact person Ophélie le Chapelain. Everyone working in a lab must know the specifics of the GMM-L material used including routes of transmission, how to handle GMM-L material in a safe way and understand the associated health hazards. The group leader/PI is responsible that the person working with viruses knows the hazards, follows the safety instructions and have been introduced how to work in the GMM-L room.

Signing up, starting work, finishing work

Sign up in the logbook every time you use the GMM-L room. You may book time in for the sterile benches in the room through the booking system. When you go into the lab always put on lab gown and gloves. Gloves and a personal lab gown are required to be worn in the room. The lab gown used in the GMM room should only be used in the GMM room and should be stored in the anteroom for the GMM room. Only approved gloves for virus work may be used (Please use the extra length orange gloves labeled EN374-2:2003 level 3, virus resistant). It is also recommended to use safety goggles to protect from accidental splashes in the eyes, especially during procedures that take place outside the microbiological safety cabinet class II (MSC II). 

The microbiological safety cabinets in the virus labs in BioClinicum are connected to the ventilation and therefore it is possible to use chemicals in these. In general, if you need to perform chemical work in a microbiological safety cabinet, please consult personnel responsible prior to your work. There should also be a label on the microbiological cabinet confirming this. Before you leave the room, take off your gloves and put them in the bag for solid waste (see below). Close both doors when going in or out of the room. The space (anteroom) between the two doors (buffer zone) should be kept clean, and should never be directly exposed to contaminated material from the GMM-L room. Always wash your hands when passing the buffer zone.

Tissue culture procedures

Follow standard cell culture procedures with the addition of the safety steps required for working in a GMM room. Work very carefully and avoid spilling. Only use tips with filter (aerosol-barrier tips). Do not use any glassware or sharps to minimize the risk of cutting. Dispose cell culture waste according to instructions (see below). After use clean the hood work surface with disinfectant for surfaces (Des +72 or Liv Des 70 or similar). Cells stored in the cell incubator should be logged on the list on the front of the incubator and plates should be clearly labeled with name and date. Cell culture consumables (plastics, such as plastic pipettes, tips, culture dishes etc.) may be stored in the GMM lab on the shelf space in a clearly labeled box. Unlabeled material left on the bench or cell incubator areas may be removed and disposed.

Lysing cells

To avoid the risk of cutting only use plastic cell culture material. Working with needles, knives, scissors or scalpels is not allowed. To store virus containing solutions or virus use the -20°C freezer in the GMM room. Virus-producing cells should be lysed to homogeneity using lysis buffer in the virus lab (no viable virus particles may leave the laboratory). Lysates from virus producing packing cells, or live packing cells, may not be taken out of the GMM room. Lysates from infected cells (non-pack cell lines) should be prepared using SDS containing lysis buffer (RIPA) or Trizol. Put cell lysates into screw-cap tubes. Spin down the lysates in the bench-top centrifuge in the GMM room. After transferring supernatants into new tubes, you may take them out of the lab. 

Transferring transduced cells out of the GMM room

Before bringing generated stable cell lines out from the GMM room they should be tested that no recombination has occurred and replication competent virus have thus not formed. To do this collect follow the protocol “Testing for replication competent virus”.

Cell counting and centrifugation 

Use the ordinary cell counting chamber to count virus-producing or virus-treated cells in the GMM room. After counting, clean the cell counting chamber with disinfection and rinse with water. When centrifuging virus-containing medium use only tubes with screw caps.

Summary

• Liquid waste should always be placed in a sealable container (conical tube, old cell media flask, white canister) before placed into the yellow waste box.
• All solid waste that can be firmly closed (e.g. cell culture flasks) should be placed directly into the yellow waste box.
• Other solid waste (e.g. plates, plastic pipettes) should be placed in the orange plastic bag marked BIOHAZARD and sealed before placing it in the yellow waste box.
• Used tips are collected in empty flasks and or white canisters placed in the yellow waste box. Do not leave consumables in hood!
• You may NOT pour any liquid waste in the sink in the GMM room or directly in the yellow waste box.
• When full, the yellow waste boxes should be properly sealed and labeled with “Smittförande avfall” and put in the recycling room for pick up. 

Solid waste

Burnable infected and non-infected material including plastic pipettes, tips, etc., should be put in plastic bag and then placed inside the yellow waste box.  This includes plastic pipettes, tips, tubes, cell culture flasks, petri dishes, gloves, tissue papers, etc., after removal of liquids. A safe way to work is to have a use the orange plastic bag marked BIOHAZARD for solid waste that can be sealed within the cabinet, which is then placed in the yellow box. All solid waste that can be firmly closed (e.g. cell culture flasks) may be placed directly into the yellow waste box. When the yellow box is filled up, close the lid and contact virus room responsible that will help you to discard the waste. Alternatively, you may dispose a full yellow waste box after you have properly sealed it and correctly labeled it as waste containing “Smittförande avfall”. Plan your work in a way to minimize the amount of waste. 

Homogenous liquid waste

Cell media, virus stocks and solutions that you do not need anymore should be collected into a sealable plastic container in the hood and then disposed into the yellow waste box.

Special cases: Hazardous chemical waste

Hazardous chemical reagents are referred from use in the GMM room. If usage is required, please consult the responsible personnel for the virus lab before use.

Glass bottles and metal containers

Used empty glass bottles and metal containers should be autoclaved and washed within the virus lab. Then they can be removed.

Lab gowns

You need to collect and wear a designated lab gown that is only used when you are inside the virus lab. The lab gown can be stored in the aft room until your work in room is completed, then it should be returned to the room for used gowns. Abandoned lab gown will be purged on a regular basis. They are changed in the usual way if they are not contaminated with spilled virus material. Take care of lab gowns contaminated with spilled virus according to the procedure described below. 

If your lab gown has been contaminated, take it off, treat the spill with Virkon, and put the lab gown in a plastic bag. Seal the bag and label it that it contains a virus contaminated lab gown. Leave the bag in the GMM room. Thereafter contact the virus room responsible as soon as possible about the contaminated gown. Thereafter it can be sent away as common laundry. 

Spill

If you have spilled virus-containing liquid on the bench, on the floor or in the microbial safety cabinet. First leave the room to avoid breading in aerosol that may have formed during the spill. Then cover the spill with absorbent, either tissues or Vermiculit. Deactivate the virus by adding Virkon on the absorbent. Instructions for how to use Virkon and preparation of Virkon (For tablets, use one tablet with 500 ml water. For bags, use one bag with 1000 ml water). Wait for a while to let the solution deactivate the virus. Throw the material used for the cleanup in a plastic bag, seal the bag and thereafter throw the bag in the yellow waste box. Never let the cleaning staff decontaminate! They do not have the expertise required.

Exposure

If you for some reason get exposed to virus we suggest following similar procedures, as you would have been exposed to blood. The description below is based on “Stick- och skärskador samt inom vården” (Puncture- and cutting related injuries including blood exposure in health care).  Please keep in mind that you should not use any sharp and cutting objects (needles, scalpels, glass pipette) when working in the room.

If something of the following occurs please follow these procedures immediately.

• If you spilled on your skin.
  • Wash first with soap and water and dry the skin
  • Wash and disinfect with 70% ethanol or an alcohol containing wash solution.
• If you splashed virus contain solution in your eye, mouth or an open wound.
  • Rinse liberally with water or physical salt solution (PBS or NaCl-solution). There is an eye washer in the room attached to the facet. Make sure you know how to use it.
• If you have a puncture related exposure (you cut yourself with an object that contain virus):
  • Do not try to press out blood from the wound.
  • Rinse liberally with water or physical salt solution (PBS or NaCl-solution).
  • Disinfect with 70% ethanol or an alcohol containing wash solution.

After you have rinsed and disinfected the wound you need to report the incident to GMM-room responsible personnel and your supervisor in a timely manner. Also, you need to file a report in KI incident reporting system (IA-system).

If you have any questions or suggestions regarding GMM-L room procedures, please contact the contact person for the GMM-L room Ophélie le Chapelain.

In case of severe major accident or pollution, injuries and similar emergencies call 112.

The general routines in BioClinicum and the Karolinska Hospital for chemical spills, personal accidents and fires apply to the virus lab as well.

In case of larger accidents, the department always has to be notified

Ola Larsson, Head of Department, work: 08-524 812 28, mobile: 073-953 9394

Chair person for work environment at OnkPat:

Paula Mannström, 070-278 2961

Responsible for work environment in BioClincium:

Magnus Nordenskjöld

Minor accidents, spills or improper use of chemicals: Contact Thomas Fritze for consultation: Thomas.fritze@stenarecycling.se , 010–445 64 81 (office hours), 070–560 75 17 (after office hours)

Also, notify Paula Mannström.

For further medical counselling, you may also contact Avonova Solna (occupational health service for KI employees), stockholmcity@avonova.se, or at 08-120 125 00  Monday to Thursday 8:00-17:00, Fridays 8:00-16:30, closed for lunch 12:00-13:00.