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Biological Safety in Laboratories

 

All experiments involving recombinant DNA, biological toxins, and other biohazards must be approved by the UH-System Biological Safety Committee.  Approval can be obtained by filling out the ​Memorandum of Understanding and Agreement Registration of Biohazardous Materials and Recombinant DNA (doc). UHD will have a standing membership on the UH-System Biological Safety Committee until such time as there is a need to establish an independent Biological Safety Committee at UHD. 

In summary, faculty working with biological hazards must complete both an MUA (and get it approved by UH) and a Project Hazard Assessment (PHA) form (docx)which must be approved by all levels at UHD.

 

 

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Biosafety Level 1

Research Labs and Teaching Labs at UHD are designated as Biosafety Level 1 (BSL-1).  BSL-1 is suitable for work involving well-characterized agents not known to consistently cause disease in immunocompetent adult humans, and present minimal potential hazard to laboratory personnel and the environment. BSL-1 laboratories are not necessarily separated from the general traffic patterns in the building. Work is typically conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is not required, but may be used as determined by appropriate risk assessment. Laboratory personnel must have specific training in the procedures conducted in the laboratory and must be supervised by a scientist with training in microbiology or a related science.

 

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General Laboratory Practices for BSL-1​

Laboratory Access

​BSL-1 facilities are restricted to those persons whose presence is required for experimental or support purposes. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in BSL-1 laboratories. All equipment necessary for conducting experiments including centrifuges, incubators, water baths, etc., shall be kept in the BSL-1 restricted access laboratory.  The PI establishes policies and procedures by which only persons advised of the potential hazards and who meet specific entry requirements (general health and training) may enter the laboratory. The PI has the final responsibility for assessing each circumstance and deciding who may enter or work in the laboratory.

Lower Containment Level Projects

Lower containment level projects may be carried out at the same time as BSL-1 projects.  However, all personnel in the area must follow BSL-1 practices and procedures.

Biohazard Signs

A universal biohazard sign with a BSL-1 designation is posted on the outside of all appropriate laboratories.  The sign identifies the biohazardous agent(s), lists the name and telephone number of the PI and CSO, and indicates the special requirements for entry into and exit out of the laboratory.

Manual

The biosafety section of this manual is designed to serve as an overview of safety for students, staff and faculty.  Personnel that are advised of special hazards are required to read and to follow specialized instructions on those practices or procedures.

Training

Laboratory personnel must receive appropriate training on the potential biohazards associated with the work. Personnel must receive annual updates, or additional training as necessary for procedural or policy changes. Training for all personnel in the area will cover these requirements and any Standard Operating Procedures.  The PI is responsible for  ensuring that, before working with organisms, all personnel demonstrate proficiency in standard microbiological practices and techniques and in the practices and operations specific to the laboratory facility.

 

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Standard Protocols for BSL-1​

The following are general guidelines for working under BSL-1 conditions.

 
  • Always minimize entry/exit to/from the room.  Plan your experiments so that once you enter the room, you stay in the room.  Bring media, cells, protocols, notebooks, etc. with you.   All manipulations involving biohazardous materials should be conducted in biological safety cabinets (biological hoods) or other physical containment devices within the laboratory.
 
  • Minimize the generation of aerosols in experimental procedures. 
 
  • Load all centrifuge bottles, carriers, and tubes in the biosafety cabinet. Wipe outer surfaces with disinfectant before transporting.
 
  • Remove and replace gloves often.    
 
  • Do not use contaminated gloves to handle common equipment.
 
  • Plastic-ware should be substituted for glassware whenever possible.  Extreme  care must be taken with any contaminated  sharp items, including needles and syringes, slides, pipettes, pipette tips, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative and must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Labs will contact the NS Stock Room when the containers are 3/4 full.
 
  • Broken glassware must not be handled directly by hand, but removed by mechanical means such as with a brush and dustpan available in the individual lab or in the NS Stock Room.
 
  • Cultures, tissues, or specimens of body fluids are placed in two containers to prevent leakage during collection, handling, processing and storage within UHD.
 
  • For shipping outside of UHD using a common carrier (i.e., Federal Express, UPS, DHL,  etc.), the PI is required to obtain any necessary permits and comply with all Department  of Transportation / International Air Transportation Association regulations.
 
  • All sterilization of contaminated materials is done in an autoclave.   Biohazardous waste must be autoclaved weekly. Autoclaves must be tested periodically for proper temperature and pressure control.  If you find that an autoclave is not operating properly, please notify the PI immediately
 
  • All research materials must be decontaminated properly.  All liquid or solid wastes must be decontaminated by autoclaving.  Reusable materials such as glass media bottles should be autoclaved after decontamination with disinfectant.
 
  • Tissue Culture dishes, plates, conical tubes and centrifuge tubes should be placed in autoclave bags.  Also, disposable pipettes, pipette tips, contaminated gloves and Kimwipes, should be placed in the autoclave bag.  All autoclave bags containing contaminated materials should be transported to the autoclave in a leak-proof container.  Once the solid waste is autoclaved, it should be lowered into another trash bag and placed in the agreed-upon area for pick up.
 
  • Mechanical pipetting devices are to be used at all times.
 
  • As is the case in all labs, there should be no eating, drinking, smoking, handling of contact lenses, or applying cosmetics while in the lab. Food must be stored outside the work area in cabinets or refrigerators designated and used for this purpose only.
 
  • Work surfaces shall be decontaminated daily and IMMEDIATELY following spills of organisms.  All spills must be cleaned up immediately. Disinfectant soap is available for spills on skin.  At the end of every working day, all work surfaces including equipment used (e.g., centrifuges and the Biological Safety Cabinet) are thoroughly wiped down with the appropriate disinfectant. 
 
  • Chairs used in laboratory work must be covered with a non-porous material that can be easily cleaned and decontaminated with appropriate disinfectant.
 
  • Spills of biological materials are decontaminated, contained and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious or potentially infectious material.  Spills and accidents that result in overt exposures to biohazardous materials are immediately reported to the PI and the NS office.  Medical evaluation, surveillance, and treatment are provided as appropriate and written records are kept. Any needle stick needs to be reported to the PI or NS office.  For more details see section below on Procedures for Handling a Spill of a Biohazard.

 

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Primary Barriers for BSL-1: Equipment

 
  • Biosafety Cabinet - Properly maintained Biosafety Cabinets (Class II or III) are used for some manipulation of biohazardous materials (e.g., pipetting, dilutions, transfer operations, plating, framing, grinding, blending, drying, sonicating, shaking, centrifuging) except where equipment design provides for containment of the potential aerosol.
 
  • Centrifuges - Use pressure seal tubes/bottles in centrifuge tube carriers.  Wipe down the outer surface of the rotor with decontaminant before taking to centrifuge. If there has been any possibility of leakage, the inner walls of the centrifuge chamber and the rotor should be immediately decontaminated.
 
  • Incubators - Use a dedicated biohazard incubator with tight fitting plugs to seal openings. The incubators in the facility are shared among several research groups, so be sure to label your containers. Clearly label any containers that contain infectious materials.
 
  • Freezers/Refrigerators – Use a dedicated storage area for biohazards.  There shall be a sign with the universal biohazard symbol on the freezer/refrigerator and materials shall be enclosed in a clearly labeled, unbreakable secondary container.
 
  • Labeling - All equipment where biohazards are used or stored must be labeled with a universal biohazard symbol and the name of the agents used or stored.

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Primary Barriers for BSL-1: Personal Protection Equipment (PPE)

 

  • BSL-1 areas require special notations with respect to PPE.

 

  • Lab Coats - Laboratory clothing that protects street clothing (dedicated cloth lab coat) must be worn in the laboratory and may not be worn outside the laboratory for any reason.  Do not store any personal items, including overcoats, hats, etc. in the BSL-1 area.

 

  • Gloves – Gloves worn while working with bacterial cultures, tissue culture cells and/or vectors.  If a spill or splatter occurs, the hand will be protected after the contaminated glove is removed. Gloves are disposed of into the autoclave bag when contaminated, and replaced frequently during procedures.  Do not wear contaminated gloves outside the work area.  Keep common areas clean.  Disposable gloves are not washed or reused.

 

  • Face Protection - Goggles, mask, face shield or other splatter guards must be used for anticipated splashes or sprays of biohazards to the face.

 

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Sample Protocol for a BSL-1 Experiment

Start-up Procedure for the Biological Safety Cabinet (Biological Hood)

 
  • Turn the blower switch to ON and before using it, let it run at least five minutes for room air to be removed from the cabinet. Make sure you have airflow by listening for blower sounds or by feeling the airflow with your fingers.
 
  • If your cabinet is equipped with a U.V. light, turn it off, and turn on the fluorescent light.
 
  • Wipe down the work surface, interior walls (not including the supply diffuser), and the interior surface of the window with appropriate disinfectant.
 
  • After placing your equipment inside the cabinet, close the sash to the proper operating height (normally eight inches but on some cabinets, 10 inches). Check the owners’ manual for proper height. Avoid working in and operating the cabinet with the sash in any other position than the manufacturers recommended sash height.
 
  • Wait another two to three minutes before working to clear all the contaminants from the work area.
 
  • Use proper PPE  as a barrier to possible exposures and to reduce contamination of your research.  Wear a lab coat, gloves, and eye or face protection, if appropriate.

Working in the Biological Safety Cabinet

 

  • Never operate a cabinet while a warning light or alarm is on.

 

  • The operator should be seated with the bottom of the sash level with his/her armpits.

 

  • Perform all work using a limited number of slow movements, as quick movements disrupt the air barrier. Try to minimize entering and exiting your arms from the cabinet, but if you need to, do it slowly and straight in/out.

 

  • To avoid excessive movements in and out of the cabinet, discard pipettes into a tray, container, or biohazard bag within the cabinet.

 

  • Keep all materials at least four inches inside the sash opening.

 

  • Plastic backed absorbent toweling can be placed on the work surface (but not on the front or rear grill openings). This toweling facilitates cleanup and reduces splatter and aerosol formation during a spill.

 

  • All materials should be placed as far back in the cabinet as is practical, toward the rear edge of the work surface and away from the front grille of the cabinet. Similarly, aerosol-generating equipment (e.g., vortex mixers, tabletop centrifuges) should be placed toward the rear of the cabinet.

 

  • The general workflow should be from clean to contaminated (dirty). Materials and supplies should be placed in such a way as to limit the movement of dirty items over clean ones. Activities that create eddy currents (opening and closing doors, personnel walking near the cabinet), should be minimized as these types of activities can disrupt the air barrier.

 

  • Open flames in a biosafety cabinet create a fire hazard and can damage the HEPA filter.  If it is absolutely necessary to use a flame, a touch-plate micro burner equipped with a pilot light or a micro-incinerator may be used. Place the burner at the rear of the work area where the air turbulence from the flame will have the least possible effect on the air stream. The burner must be turned off when the work is completed.

 

  • Aspirator bottles or suction flasks should be connected to an overflow collection flask containing appropriate disinfectant, and to an in-line HEPA or equivalent filter.

Completing the Work in the Biological Safety Cabinet

 
  • All equipment that has come in contact with a biological agent should be decontaminated. The cabinet should be allowed to run for at least three minutes with no activity so the airborne contaminants will be purged from the work area. Disinfect the surface of the equipment prior to removing it from the cabinet.
 
  • After all items have been removed, wipe down all surfaces of the cabinet with a disinfectant, including the sides and back, and the interior of the glass.

Biological Safety Cabinet Certification   

The cabinet must be certified upon installation, annually thereafter, and any time it is moved (even if just across the room).

 

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Common Errors to Avoid in the Biological Safety Cabinet

 
  • Keep papers, paper towels, plastic backed absorbent toweling, vials, or any other objects from being pulled into the back, front, or side slots if no grill is in place to prevent this.
 
  • Do not store equipment or supplies in the cabinet.
 
  • Do not use the top of the cabinet for storage. The HEPA filter could be damaged and the airflow disrupted.
 
  • Do not place items on the front or rear perforated grills. This reduces the airflow.
 
  • Make sure the cabinet is level. If the cabinet is not level, the airflow can be affected.
 
  • Never disengage the alarm, as it indicates improper airflow, thereby affecting performance and endangering the researcher or the experiment.
 
  • Never completely close the window sash with the motor running. This may cause the motor to burn out.

 

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Additional Information on Cell Culture

At this time only cell cultures approved for work in BSL-1 laboratories may be utilized at UHD.  The CSO will require PIs to provide verification that the cell lines they are currently working with can be handled in BSL-1 facilities.

Storage and retrieval of frozen cell cultures from liquid nitrogen requires appropriate PPE. The three major risks associated with liquid nitrogen (-196° C) are: frostbite, asphyxiation, and exposure. Gloves thick enough to act as insulation but flexible enough to allow manipulation of ampoules should be worn. When liquid nitrogen boils off during routine use of the freezer, regular ventilation is sufficient to remove excess nitrogen, but when nitrogen is being dispensed, or a lot of material is being inserted into the freezer, extra ventilation will be necessary.

 

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Procedures for Handling a Spill of a Biohazard​

There are currently two types of cultures that are utilized at UHD which could be classified as a biohazard.  The culture types and the procedures for disinfecting them can be seen in Table 1 (below).

Biohazard     Agent(s) Type of Disinfectant Disinfectant Concentration Disinfectant Procedure

Means of Disposal

Eukaryotic cell cultures Bleach (sodium hypochlorite) 2% Bleach before autoclave Autoclave or dispose of in the Biohazard waste
Unused bacterial cultures Bleach 2% Bleach before autoclave Autoclave or dispose of in the Biohazard waste

Table 1.  List of all potentially biohazardous materials utilized at UHD as of 2012.

Personal Contamination

 
  • If a spill of biohazardous agents (cultures) occurs on a person in the lab, he/she should remove the contaminated articles or wash the exposed area with soap and water for at least one minute. 
 
  • If eye exposure occurs the eye wash station should be used per instructions. Medical attention should be sought as appropriate. 
 
  • If there is an injury or a near miss of an injury a Report of Safety/Health Hazard and Near Miss must be completed in NS (room N813 in the One Main Building).  

Spills Inside the Biosafety Cabinet

 
  • Move the glass shield down and wait at least five minutes to allow the Biosafety Cabinet to filter and clear aerosols.
 
  • Wearing appropriate PPE (at a minimum lab coat, safety glasses and gloves), apply disinfectant for a minimum of 20 minutes contact time directly on the spill and on all  potentially exposed surfaces of the cabinet.
 
  • Wipe up spill with disinfectant-soaked towels or other appropriate absorbent material.  Wipe the walls, work surfaces, inside of sash and any potentially contaminated equipment with disinfectant soaked towels before removing it from the Biosafety Cabinet.
 
  • Discard contaminated disposable material using appropriate biohazardous waste disposal procedures.  Wipe down contaminated reusable items with disinfectant then place in autoclave bag or autoclave pans with lids for autoclaving.
 
  • Those items that are non-autoclavable should be wiped down with disinfectant and kept wet for a minimum of 20 minutes before removal from Biosafety Cabinet.
 
  • Remove protective clothing and when finished doing that  and place in biohazard bag for disposal or autoclaving for reusable items.
 
  • Run the Biosafety Cabinet for 10 minutes after clean-up before reusing.
 
  • WASH HANDS!

Surface Spill in the Lab Outside the Biosafety Cabinet

 
  • Be sure you are wearing appropriate PPE for size and nature of the spill, including at a minimum: lab coat, gloves and safety glasses.
 
  • Place dry paper towels to establish a physical barrier between the spill and yourself. Then layer a second set of disinfectant-soaked towels over the spill.
 
  • Starting from the outside and working in, carefully soak the spill with disinfectant being careful to minimize aerosolization.
 
  • Decontaminate all items within the spill area.  Wait at least 20 minutes for disinfectant contact time to allow for adequate inactivation.
 
  • Wipe equipment and reusable items with the disinfectant.
 
  • Wipe up spill and discard contaminated disposables in the biohazard waste container.
 
  • If sharps are present, use a mechanical device such as a dust pan and brush to pick up the sharps and place in an approved sharps container.

Spill Inside a Centrifuge

Wipe rotors and buckets with 70% ethanol, 1% SDS or 1% bleach.

Thoroughly disinfect inside of centrifuge with a minimum contact time of 20 minutes using a disinfectant that follows manufacturer’s recommendations and is effective for spilled agent.  Dispose of contaminated materials in the biohazard waste container.

Spill Outside the Lab (in Transit)

Should a spill occur in a public area, do not attempt to clean up without the appropriate PPE.

Secure the area around the spill.  Contact NS the CSO or your PI.

Reporting Requirements

The release of a large volume of organisms (as a general rule anything over 10 ml) or a spill of material containing a high concentration of organisms must be reported to the PI or the CSO immediately.  The PI and the CSO will assess the risk and initiate an emergency response with EHS as requested/appropriate.  In the case of exposure a Report of Safety/Health Hazard & Near Miss form must be filled out.  For students a Student/Visitor Accident Report Form will also have to be filled out by the PI or Chief Safety Officer. 

 

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Last updated 7/22/2016 6:05 AM