BIOSAFETY LEVEL 2 PROTOCOLS BIOPROCESS OPTIMIZATION LABORATORY - POULIOT BUILDING, ROOM 1541 ALAIN GARNIER JUNE 2007, LAVAL UNIVERSITY

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1 BIOSAFETY LEVEL 2 PROTOCOLS BIOPROCESS OPTIMIZATION LABORATORY - POULIOT BUILDING, ROOM 1541 ALAIN GARNIER JUNE 2007, LAVAL UNIVERSITY

2 2 Preamble This document describes the procedures to follow for safely handling BSL 2 agents under study in the Bioprocess Optimization Laboratory (BOL). The laboratory reserved for these activities is located in room #1541 of the Adrien- Pouliot building. For further security, these regulations will be applied for all microorganisms handled in this laboratory. The document starts with a list of the sources to consult to complete an adequate theoretical biosafety training. This training will be completed by an observation period in the laboratory. The last page of the document allows the conservation of a file on the training of all the staff of the laboratory. It should be filled and signed before being allowed to work autonomously in the laboratory.

3 3 Emergencies Personnel to contact in cases of emergency Localized minor spills: Lab supervisor Coordinates Major spills (in litres): Lab supervisor Coordinates Prevention and security service Emergencies Assistance See section 5 of the present document: Spills, incidents and emergencies.

4 4 Table of contents Preamble...2 Emergencies List of documents to read and consult General and BSL2 measures Use of biological safety cabinets (BSC) Decontamination and elimination of biological wastes Spills, incidents and emergencies Registration of conserved specimens, used and destructed Material safety data sheets of level 2 organisms handled in lab Consent form...12

5 5 1- List of documents to read and consult - This document «BIOSAFETY LEVEL 2 PROTOCOLS». - The «Laboratory biosafety guidelines», Public health agency of Canada: - The «Containment Standards for Veterinary Facilities», Canadian Food Inspection Agency:

6 6 2- General and BSL2 measures - Consult the material safety data sheets of the pathogenic agents to be manipulated before starting the work. - Access to laboratory is limited to authorized personnel. - Personal items such as purses and outdoor clothing should be kept in the change room or the personnel s office. - Eating, drinking, smoking, storing of either food, personal belongings, or utensils is not permitted in the laboratory. - Applying cosmetics, and inserting or removing contact lenses is not permitted in the laboratory. - Wearing jewellery is not permitted and long hair is to be tied back or restrained so that it cannot come into contact with hands, specimens, containers or equipment. - Open wounds, cuts, scratches and grazes should be covered with waterproof dressings. - Protective laboratory clothing, properly fastened, must be worn by all personnel (lab coat), protective laboratory clothing must not be worn in non laboratory areas; laboratory clothing must not be stored in contact with street clothing. - Suitable footwear with closed toes and heels must be worn in all laboratory areas. - Wearing protective eye glasses and face shields (goggles) if necessary to guard the eyes and face against risks of liquid projections and objects impact whether during routine operations or in exceptional cases. - Gloves must be worn for all procedures that might involve direct skin contact with biohazardous materials. - Gloves and dedicated laboratory clothing must be removed before leaving the laboratory. - Oral pipetting of any substance is prohibited in the laboratory.

7 7 - If a known or suspected exposure occurs, contaminated clothing must be decontaminated before laundering. - The use of needles, syringes and other sharp objects should be strictly limited. - Needles should not be bent, sheared, recapped or removed from the syringe. - Hands must be washed 1) on entering the laboratory, 2) after gloves have been removed, 3) before leaving the laboratory and 4) at any time after handling materials known or suspected to be contaminated. - The laboratory is to be kept clean and tidy. - Doors to the laboratory must not be left open. - Do not hang street clothing inside the laboratory, leave these clothing in the change room or in the office. - All the procedures that may produce infectious aerosols and that involve high concentrations or large volumes of biohazardous materials must be performed in the biological safety cabinets BSC (biological hood), see point 3 «Use of the biological safety cabinets». - Disinfectants effective and appropriate against the agents handled must be available at all times in the laboratory and used for decontaminating all surfaces and materials. - All contaminated materials, solid or liquid, should be decontaminated before disposal or reuse. These materials must be contained in such a way as to prevent the release of the contaminated contents during removal see point 4 «Decontamination and elimination of biological wastes». - Leak-proof containers are to be used for the transport of infectious materials within facilities. - Accidents of exposures to infectious materials and losses of containment must be reported immediately to the laboratory supervisor. - Annual vaccination against influenza should be carried out (contact working health and security section of Laval University, phone 2110).

8 8 3- Use of Biological Safety Cabinets (BSC) - Use a BSC for procedures with potential for producing infectious aerosols (removal or addition of liquids containing infectious agents, opening of tubes after centrifugation, etc). - Use the BSC for the procedures that involve high concentrations or large volumes of biohazardous materials. - Do not use the UV lights of the BSC. - Before using the BSC 1) place the sash in the appropriate position if necessary, 2) check the air intake and exhaust grilles for obstructions, 3) confirm inward airflow either by checking the counters or by holding a tissue at the middle of the edge of the viewing panel and ensuring that it is drawn in, and 4) disinfect the interior surfaces with a suitable, noncorrosive disinfectant. - During handling inside the BSC 1) avoid excessive movement of hands and arms through the front access opening, 2) ensure not to obstruct the air grilles, 3) keep discarded, contaminated material to the rear of the cabinet separated from sterile clean solutions and instruments, 4) perform operations as far to the rear of the work area as possible, 5) avoid performing operations over the biological material, and 6) ensure to discard wastes in containers inside of the BSC (sharp objects such as pipettes should be placed in a secure container). - Do not work with open flames inside the BSC. - At the end of BSC use 1) allow the BSC to function for 5 minutes, 2) close or cover open containers before removing them from the BSC, 3) surface disinfect objects in contact with contaminated material before removing them from the BSC, 4) disinfect the working surfaces while the BSC is in run, 5) remove the gloves and dispose of them inside the BSC, and 6) ensure that all biological materials are disposed safely.

9 9 4- Decontamination and elimination of biological wastes - Ensure that the disinfectants are appropriate and effective against the handled agents, consult the material safety data sheets, section IV, for each of the agents on the website of the Public health agency of Canada, on the following link - Desinfectant solutions such as sodium hypochlorite lose their germicidal action with time, it is recommended to use a freshly prepared solution each day. - Decontaminate all the liquid contaminated materials with a disinfectant appropriate for the pathogenic agent in concern, such as a solution of sodium hypochlorite (chlorine or bleach) at a final concentration of 1% for a minimum of 12 hours before disposal. - Place the solid contaminated wastes, already in an autoclavable container, in the yellow tank. Keep the cover of the tank closed. - When the yellow tank is filled, contact the biological waste management center for pick up. - Decontaminate the contaminated or potentially contaminated surfaces with a disinfectant appropriate for the pathogenic agent in concern, such as a solution of ethyl alcohol or isopropyl alcohol 70% (cells, retroviruses, and influenza) or Bioclean 2 (adenoviruses). Leave the surface in contact with the solution for 10 minutes before rinsing and wiping. In the case of decontamination with alcohol, rinse first the surfaces with a humid buffer and consider this buffer as a solid contaminated material. - For the bioreactors: empty and rinse under containment (collect and decontaminate the liquids). Decontaminate at warm temperature (60ºC, 1 hour minimum). - Place the needles, syringes, and other sharp objects in puncture resistant containers such as the yellow containers for needle and blade waste.

10 10 When the container is filled, contact the biological waste management center, for pick up. - Place all the contaminated materials, solid or liquid, in containers preventing their release if they should be removed from the laboratory. - Decontaminate the surfaces and the benches at the end of each working session or after each spill of infectious materials. 5- Spills, incidents and emergencies Localized minor spills Necessary material Protective equipment: - Overshoes - Rubber gloves - Face and eye protection: (e.g., glasses, goggles, face shields) - N95 Mask - Lab coat Decontamination kit: - Chlorine (bleach) 1% or 5,25% (non diluted) - Paper towels - Tongs or any other instrument to avoid cuts or punctures - Decontamination bags Procedure 1. Contact the lab supervisor. 2. Put the protective equipment. 3. Absorb the spill with paper towel. 4. Apply the appropriate disinfectant from the outside to the inside. 5. Leave in contact for 30 minutes. 6. Clear away the materials; if glass fragments, use tongs. 7. Place in a bag for decontamination. 8. Repeat steps from 3 to Place all the materials (equipment and kit) in a bag for decontamination. 10. Sterilize in the autoclave. 11. Wash the hands and the exposed skin with water and soap, all the surfaces should be thoroughly lathered with soap, using friction for a minimum duration of 20 seconds, rinse, dry; if Foot-

11 11 or elbow-operated faucets are not available, turn off the faucet handles avoiding recontaminating washed hands. Major spills (litres) 1. Evacuate the laboratory and close the door. 2. Prevent any circulation within the contaminated area. 3. Contact the lab supervisor and the local security and prevention service. Provide your coordinates: a. Name b. Phone number c. Building d. Department e. Room number f. Type of hazardous material spilled g. Quantity 4. Remove the contaminated clothing in such a way which keeps the contamination to the inside; place them in a bag for decontamination. 5. Wash hands and the exposed skin with water and soap, all the surfaces should be thoroughly lathered with soap, using friction for a minimum duration of 20 seconds, rinse, dry; if Foot- or elbow-operated faucets are not available, turn off the faucet handles avoiding recontaminating washed hands. 6. Remain in proximity of the spilled area to assist the decontamination team. (Inspired from Procedures in case of spill of infectious substances, C Noël, SSP, U Laval) 6- Registration of conserved specimens, used and destructed - Initialize and date all reagents entering the laboratory.

12 12 - Register all the specimens of biological material in the appropriate document. 7- Material data sheets of BSL2 organisms - Influenza virus, technical data sheet safety-security - infectious substances, Office of laboratory security, Public health agency of Canada, 2005 (appendix) 8- Consent form I acknowledge having read and understood this document as well as the recommended material in section 1. I commit to follow the protocols as they are here presented. Name: Location: Signature: On (yy/mm/dd):