Environmental Health and Safety

Revised: January 22, 2020

Laboratory Safety Training

Outline

• Background and Purpose
• Housekeeping and Fire Safety
• Chemical Safety
• Biological Safety
• Laboratory Security
  
• Radiation and Laser Safety
• Personal Protective Equipment (PPE)
• Emergency Eyewashes and Showers
• Compressed Gas Cylinder and Cryogenic Safety

Background and Purpose

This training is required every two years for all personnel who work in laboratories. It provides laboratory safety fundamentals, and shall be augmented by lab-specific training that addresses hazards specific to the lab. Note that the Biological Safety Training module has been eliminated and that module has been incorporated into this training.

Principal Investigators should contact the Laboratory Safety Officer shortly after reporting onboard so that an initial laboratory safety inspection can be completed using the checklist contained in the Laboratory Inspection Program.

When a Principal Investigator leaves the Health Sciences Center they must follow the Lab Closeout Policy so that “vacated research space is thoroughly cleaned and prepared for reassignment to another investigator or unit.”

Housekeeping and Fire Safety

 

Housekeeping

• Eating or drinking is not allowed in laboratories that contain chemical, biological or radioactive materials.
• Dispose of trash when it is generated in appropriate containers. Prevent accumulation of trash within the laboratory.
• Don't place empty bottles and other trip hazards in a walkway.
• Keep chemicals and glassware away from the edge of counters.
• Immediately clean up all spills.

Fire Safety

• Know the location of the nearest fire extinguisher.Ensure you have the equipment to address any special chemicals (e.g., pyrophoric or water-reactive chemicals).

• Flammable cabinet required if > 10 gals of any flammable present.

• Secure compressed gas cylinders.

• Maintain at least 18 inches of clearance between fire sprinkler heads and any other material.

• Ensure electrical wiring is free of cuts and not fraying.
• In the event of an alarm, secure any procedures that left unattended will pose a hazard and evacuate.

Chemical Safety

Hazardous Communication (Hazcom)

The Hazardous Communications Program ensures that the hazards of all chemicals used at LSUHSC are evaluated and that information concerning their hazards is transmitted to all applicable personnel.

A key element of the program is the Safety Data Sheet (SDS), which are used to familiarize the user with a chemical's:

• Chemical and physical properties.
• Toxicity and Health Effects.
• Compatibility, Safe Handling and Storage.
• Spill and Fire response.

SDSs must be readily available, either in hard copy or on a computer hard drive as long as employees have immediate access without leaving the work area.
Ensure a backup is available for rapid access in case of a power outage.

Chemical Ordering, Storage, and Inventory

• When ordering chemicals, always order only what is needed; a six month supply is plenty.
• In chemical storage areas:
• Maintain chemical spill kits and fire extinguishers.
• Provide adequate shelving. Don't store on the floor.
• Store quantities of 10 gallons or more of flammable materials in an approved Underwriters Laboratory (UL) or Factory Mutual approved flammable storage cabinet.
• If cold storage is required for flammables, use a refrigerator that is UL approved and rated for flammable storage.
• Maintain an inventory of all chemicals per the Chemical Procurement, Inventory and Security Policy.  As a minimum, you must update the inventory review statement using the On Site program at least every 12 months.
  

Chemical Incompatibilities

• To avoid interaction between incompatible chemicals, all chemicals should be separated into compatible hazard groups, then placed alphabetically within each group.
• Since many chemicals present multiple hazards, consult the SDS to determine the “primary” hazard class of a chemical.
• For more information on chemical storage, see Chemical Ordering and Storage Procedures.

Peroxide Forming Chemicals

A wide variety of organic compounds can spontaneously form peroxides on exposure to air. Peroxides are sensitive to heat, friction, and shock.

• Label containers with receiving, opening and disposal dates.
• Store away from heat sources and sunlight.
• Do not attempt to open a container or a peroxide forming compound if there are whitish crystals around the cap and/or in the bottle because it may be potentially explosive.
• For additional information on peroxide forming chemicals see Standard Operating Procedure for Safe Use and Handling of Peroxide Forming Chemicals.

Engineering Controls - Fume Hoods

Chemical Fume Hoods are an important tool used to minimize exposure.

• Work with volatile chemicals in fume hoods when feasible.
• Fume hoods are not a place to store chemicals or equipment.
• Fume hoods are not designed to collect particulates and are not HEPA filtered. They are not to be used with infectious or potentially infectious materials.
• If your hood is not functioning properly, create an On-line Service request to have it repaired.

Prior to Using a Fume Hood

• Check the hood certification sticker on the front of the hood to ensure that the air flow is between 80 and 150 feet per minute when tested. Fume hoods are inspected and certified annually by EH&S.
• Check visual alarms to ensure that the hood is functioning properly.
• Hold a Kimwipe or tissue at the face of the hood to ensure air is flowing into the hood when the fume hood is turned on.
• Check baffles to be sure slots are open and unobstructed by equipment or containers.
• Ensure that all chemicals and equipment are at least six inches behind sash.
• Decrease turbulence by opening / closing sash slowly; avoid swift movements inside / outside of the hood.
• Keep sash as low as possible (closed when not in use).
• For more information, see the Standard Operating Procedures for Safe Operation of Chemical Fume Hoods.

Chemical - Waste Disposal

• No chemical waste shall be discarded via the sink to the sanitary sewer system.
• When collecting waste, compatible waste streams may be collected in one container. Never mix incompatible wastes.
• Chemical disposal containers:
• Use containers that are in good condition and are compatible with waste storage type.
• Always keep the container closed during storage; never leave a waste container open with a funnel in it.
• Use containers with capacities of one gallon or less and never over fill them.
• Label all waste containers with:
• The words "Hazardous Waste".
• Principle chemical constituents.
• Date that waste was first placed in the container.

• To dispose of batteries:
• Place in individual plastic bags or cardboard boxes for collection.
• Cover terminals with tape if not placed in individual bags or boxes.
• For disposal of mercury containing equipment, place all equipment containing elemental mercury in a plastic bag and seal.
• Waste Pick-up:
• Submit an on-line service request for every hazardous chemical waste pick-up.
• For additional information, review the Chemical Waste Management Procedures.

Chemical - Spill Response

• Regardless of volume spilled, do not attempt to clean up a spill if the chemical is unknown or if known chemical is extremely toxic. If you are unable to safely clean the spill on your own, contact University Police at 568-8999. University Police will then notify Environmental Health and Safety to respond.
• See the Chemical Spill Response Policy and Procedures for more information.
• If you need assistance cleaning the spill, contact University Police at 568-8999, and they will contact Environmental Health and Safety to respond.

Biological Safety

Biohazards and Exposure

Biosafety is the application of combining laboratory practices, procedures, facilities, and safety equipment to protect human health and prevent release of biological materials into the environment.

A biohazard is an agent of biological origin that has the capacity to produce deleterious effects in humans, such as microorganisms, toxins and allergens derived from those microorganisms, or higher plants and animals.

Examples include:

             

Exposures are typically the result of needle sticks; accidental exposure to eyes, mouth and mucous membranes; bioaerosols; and spills.

Biological Materials Inventory and Storage

Maintain an inventory of all biological materials per the Biological Materials Inventory and Control Policy.

As a minimum, you must update the inventory review statements using the On Site program at least every 12 months. Personnel who ship biological materials must complete the Shipping Biological Materials Training or equivalent dangerous goods training every two years.

Risk Assessment

Work practices, use of containment equipment, PPE, training, etc. should be guided by a thorough risk assessment (RA).

A risk assessment ensures protection of personnel, the environment, the community and the integrity of your experiments.

PIs are responsible for conducting the RA and should include the RA in lab-specific training and biosafety manuals.

Risk assessment guidance can be found in the CDC BMBL 5th Ed.

RAs are comprised of four steps:

1. Identification of health hazard
2. Quantification of the hazard
3. Exposure assessment
4. The probability of disease

And should include consideration of:

• Virulence
• Pathogenicity
• Infectious dose
• Environmental stability
• Route of spread
• Communicability
• Operations and manipulation
• Quantity and availability of vaccine or treatment

rDNA

All rDNA used on campus is considered a biohazard. Experiments involving rDNA require specific biological barriers.

Barriers should limit (i) the infectivity of a vector or vehicle – plasmid or virus – for specific hosts, (ii) its dissemination and survival in the environment.

Newer-generation vectors are designed to decrease the probability of dissemination of rDNA outside of the lab and are preferred.

rDNA should be a part of the comprehensive risk assessment described in IBC documentation for your lab, taking into account source, vector, polypeptide or gene product, etc.

Biological Safety Biosafety Levels (BSLs) Work Practices

BSL determination should be informed by risk assessment which is based on the risk group (RG) of the microorganisms present in the lab.

---> Infectious Microorganisms by Risk Group (Increasing Risk) --->
A/BSL-1	A/BSL-2	A/BSL-3	A/BSL-4
Low individual risk (non-infectious to healthy adults) Low risk to community	Moderate individual risk (Not generally severe, treatment usually available) Low risk to community	High individual risk (Treatment may or may not be available) Low risk to community	Severe individual risk (Treatment often not available) High risk to community
Examples
E. Coli lab strains (e.g., DH5a, K12), Mice, Rats, Rabbits	Human cells, fluids, tissues, NHP cells, fluids, tissues, Lentiviral vectors, Rhesus Macaques, Toxins with an LD50 >100 ng/mg, Animals infected with BSL2 agents	M. tuberculosis, West Nile virus, Francisella tularensis, Yellow fever virus, Monkeypox virus, Animals infected with BSL3 agents	Ebola virus, Lassa virus, Marburg virus, Animals infected with BSL4 agents

PPE recommendation and guidance are available for each BSL

---> Biosafety Level Precautions (Increasing Risk) --->
A/BSL-1	A/BSL-2	A/BSL-3	A/BSL-4
Lab coats, gowns, etc., Eye protection, Latex or nitrile gloves	Lab coats, gowns, etc., Eye protection, Latex or nitrile gloves, Change when contaminated, Double glove when necessary, Remove gloves and wash hands after working, Do not re-use gloves	All manipulations performed inside a BSC, Full protective clothing that must not leave the lab, Eye protection, Latex or nitrile gloves, BSL-3 work practices, Appropriate respiratory protection	All PPE indicated up to and including BSL-3 Positive-pressure suit, Special facility engineering features

A risk assessment pertinent to your lab and your work should yield PPE and work practice directives specific to your workplace.

(Click or tap image for expanded view)

BSL-1 Facility Design

• Laboratory has doors to limit traffic.
• Hand washing sink is available.
• Work surfaces are easy to disinfect.

BSL-1 Work practices

• Limit access when working.
• No eating, drinking, applying cosmetics or handling contact lenses
• No mouth pipetting.
• Gloves must be worn and lab coats and protective eyewear are recommended.
• Minimize splashes and creation of aerosols.
• Disinfect waste and work surfaces.
• Biological waste should be placed in a biohazard disposal box, labeled, and placed outside for pickup when ¾ full.

(Click or tap image for expanded view)

BSL-2 Facility Design

All BSL-1 requirements, plus:

• Autoclave is available.
• Eyewash is present.
• Signage is posted.
• Biological waste stream is separate.

BSL-2 Work Practices

All BSL-1 practices, plus:

• Ensure the doors to the BSL2 area are closed while BSL2 research is being conducted.
  
• A supervisor must limit access to those who are trained and approved.
• Policy for handling sharps must be implemented.
• Laboratory equipment must be routinely decontaminated.
• Protective lab coats or disposable gowns must be worn.
• Laboratory-specific Biosafety manual must be available in the lab.

Hand Washing

• All laboratories are required to have a sink available for hand washing.
• Wash hands for 15 seconds using warm water and mild – preferably liquid – soap. Rinse with warm running water.
• Dry with disposable paper towel.
• Alcohol-based hand sanitizers are an alternative to hand washing.
• Sanitizers are effective against common clinical microbes, but have not been tested against laboratory pathogens.
• Hand washing is preferred over hand sanitizers.

   

Disinfection

• Disinfection is the process of reducing a contaminant load.
• Can be accomplished in the laboratory using a 70% solution of ethanol (EtOH) or a 10% solution of bleach. (sodium hypochlorite).
• All works surfaces and materials should be disinfected before and after use.
• SOPs for routine decontamination are available on the Biological Safety page of the EH&S website.

Autoclave Decontamination

• Place items in a secondary container made of stainless steel or autoclaveable plastic.
• Most pathogens and recombinant molecules are sensitive to temperatures above 121ºC for after 20 or more minutes.
• Larger loads require more time and should be arranged in a way that allows for steam penetration (i.e. not too densely packed).
• Do not cap vessels or add excessive liquid to the load.
• Use caution when opening autoclave at the end of the cycle – steam is usually still in the chamber.
• See Autoclave SOP for additional information.

     

Safe Handling of Liquids

Liquid biohazard materials (cultures, blood or body fluids) must be placed in a container with a lid to prevent leaks and spills during collection, handling, processing, storage, transport and shipping.

   

Aqueous biological materials such as blood, cell cultures or microbial cultures must be either:

• Decontaminated with bleach by adding 1 part bleach to each 9 parts liquid waste. Let stand for at least 60 minutes.

OR

• Decontaminated by autoclaving on liquid cycle. Do not autoclave bleach- treated liquid waste.

Biological Safety Equipment

Biological Safety Cabinet

Biological Safety Cabinets (BSC) are an important tool used to control bioaerosols and minimize exposure.

• BSCs are designed to protect the product, worker, and environment. Laminar flow hoods only protect the product.
• BSCs remove particulates, not chemical vapors.
• All Biosafety cabinets must be certified annually, prior to initial usage, and if moved.
• At BSL2 and above, any procedure that may produce aerosols must be performed inside of a Biological Safety Cabinet.
• Contact EH&S before purchasing a new cabinet for help in selecting the appropriate cabinet.
• For more information, see the Standard Operating Procedures for Safe Operation of Biological Safety Cabinets.

• Uses High Efficiency Particulate Air (HEPA) filters.
• Does not protect against vapor or fumes, which may damage HEPA filters.
  • Class I: Inward airflow protects personnel. Exhausts to outside.
  • Class II:
    • Four different types.
    • Protects personnel, materials and environment with directional airflow and multiple HEPA filters, as pictured.
  • Class III: Both inlet and exhaust air are HEPA filtered.

Safe Operation of BSCs – before use

• Disinfect cabinets before and after each use with 70% ethanol or 10% bleach solution.
• After disinfecting the cabinet, load supplies and allow the cabinet to run for 10-15 minutes before beginning work.
• Supplies should include a small autoclave bag, sharps container and beaker with disinfectant for liquid waste.
• Your BSC should have a current certification label. Contact EH&S if certification or maintenance is needed.

• Check inward airflow by holding a piece of tissue near the opened sash.
• Segregate clean and dirty materials to avoid contamination.
• Place materials 4” or more inside cabinet to avoid disrupting airflow.
• When reaching into the cabinet to work, avoid abrupt or excessive movements to maintain airflow.

Safe Operation of BSCs – after use

• When finished working, discard solid and liquid waste and sharps in the appropriate manner.
• Disinfect the work surface.
• Lower the sash.
• Turn on UV light.

Using BSCs with Radiation or Chemicals

Working with Radiation

• Radiolabeling of biological samples must be done inside a BSC.
• The BSC must be labeled with a Radiation Warning label.
• Proper shielding must be in place inside the BSC.

Working with Chemicals

• Some BSCs allow for work with non-volatile chemicals – some have restrictions on the quantity to be used.
• Check the indications for your particular class and type of BSC.

More information can be found at the Standard Operating Procedure for the Safe Operation of Biological Safety Cabinets and NIH YouTube video.

Laminar Flow Hood

• Is not a BSC and does not provide personnel protection.
• Typically used for nucleic acid manipulation or other procedures that are very sensitive to contamination, but that do not pose a risk to personnel.
• Air flows out toward the user.
• Not to be used for work with infectious or potentially infectious materials.

Safe Operation of Centrifuges

• Check tubes for cracks, leaks or chips.
• Use matching sets of tubes and buckets to ensure that the centrifuge is properly balanced.
• Check that tubes and cups are sealed and that the rotor is locked and buckets are properly seated
• Close lid firmly.
• When the cycle is finished, allow the rotor to come to a complete stop before opening lid.

Biological Safety - Waste Disposal, Spill Response and Biosecurity

Waste Disposal

Biohazardous waste includes the following general categories:

• Cultures and stocks.
• Pathological waste.
• Human and animal blood and blood products.
• Used and unused sharps (e.g., needles, syringes, pipettes, scalpels, broken glass, slides, cover slips).
• Animal waste (e.g., carcasses and bedding).
• Isolation wastes. Any other refuse, which has been mingled with any of the waste(s) listed above.

Never place chemicals, chemical bottles, radioactive materials, or other trash in biowaste containers.

Glass Disposal

• Uncontaminated or decontaminated glass may be disposed of in a designated, labeled cardboard box.
• Box should be sturdy and in good condition.
• Take care not to overload the box – it should be kept to a reasonable weight, approximately 25 lbs.

Sharps Disposal

• Used disposable needles must not be bent, altered, broken, recapped, removed from disposable syringes, or otherwise modified.
• Always dispose of contaminated sharps in an approved, puncture-resistant sharps container.



• Dispose of container when it is ¾ full by sealing the container and placing in a biological waste box.
• See the Standard Operating Procedure for the Safe Handling of Sharps for more information.

Bioboxes

To be used for:

• All items contaminated with human or animal blood, fluid or tissue.
• Stocks, cultures or waste from infectious materials or microorganisms.
• All materials that may be contaminated with recombinant molecules.

Do not place sharps directly into the biological waste box. Place sealed sharps containers in a biological waste box.

When box is ¾ full or reaches 25 lbs. close and tie liner, securely close lid, label with PI name and room number and place in hallway for pickup.

Spills and Potential Exposures

Report all incidents involving potential exposure to rDNA to the Biological Safety Officer immediately. If the spill occurs after hours notify University Police. Depending on the type of incident and the level of exposure, notification of the NIH may be required within 24 hours of the incident.

Response to spills depends on:

• Agent identity and associated risks.
• Agent's Biosafety level.
• Amount and location of material spilled.

If you need assistance to safely clean the spill then contact University Police at 568-8999. University Police will then contact Environmental Health and Safety to respond or see the Biological and rDNA Spill Response Procedures for more information.

Laboratory Security

In order to ensure the security of potentially harmful biological materials:

• Carefully balance security, safety and the effective conduct of research while perfoming your operations, to include the issue of closing laboratory doors. Due to the nature of research, it is not always adviseable due to safety and efficiency issues to close doors to a laboratory even if that laboratory is temporarily unoccupied. However, in general, close doors of all laboratories when labs are unoccupied for an extended period (e.g., leave for lunch, end of day, attend a meeting)
  
• Control access to areas where biological agents and chemicals are stored.
• Know who is in your work area.
• Know what materials are being brought into and taken out of your laboratory.
• Have a protocol in place for reporting incidents or suspicious activities or people.
• All labs must have a CAUTION placard affixed to the door. The placard will include the name and off campus phone number of the Principal Investigator and the assigned point of contact, and the appropriate hazard labels, (e.g., Global Harmonized System (GHS) hazard labels, biohazard label and radiation trefoil hazard label). If you need additional CAUTION placards or hazard labels, contact EH&S and they will be delivered to your laboratory.
  

Radiation and Laser Safety

Approval for Use of Radiation Sources

Prior to beginning work with any radiation source, the PI must submit the Radiochemical Use Application Form to the Radiation Safety Committee for approval. Furthermore, personnel must complete Radiation Safety Training available from the Radiation Safety Officer (RSO).

Therefore, if you intend on using radiation sources, please contact the RSO to schedule training.

For additional information see the LSUHSC Radiation Safety Committee Charter.

Approval for Use of Lasers

Prior to beginning work with any category 3b or 4 laser, lasers must be registered with the Radiation Safety Officer.

Additionally, “on-line” Laser Safety training must be completed.

Therefore, if you intend on using class 3b or 4 lasers, please contact the RSO.

For additional information see the LSUHSC Laser Safety webpage.

Personal Protective Equipment

Do not wear shorts or any clothing that exposes any skin other than arms or face.

Do wear the appropriate Personal Protective Equipment (PPE) for the situation:

• Gloves.
• Splash Goggles / Face Shields.
• Lab Coats.
• Respirators.

Provide proper storage of all PPE.

See the Laboratory Attire and PPE and Personal Protective Equipment policy web pages for more information.

Lab Coats and Gowns

Lab coats or disposable gowns should be worn over street clothes any time you handle hazardous materials.

The type of covering necessary and the frequency of changing is specific to your work and should be indicated in lab-specific training.

Lab coats and gowns must never be worn outside of the work area.

See the Lab Coat Use, Selection and Cleaning web page for more information.

Eye and Face Protection

Wear protective eyewear when conducting procedures that have the potential to create splashes of microorganisms or other hazardous materials.

People who wear contact lenses should also wear eye protection.

At BSL2 and above, eye and face protection must be used for anticipated splashes and sprays of infectious materials when the microorganism is handled outside of a biosafety cabinet or other containment device.

Respiratory Protection

Respirators must be worn when there is potential exposure to contaminants such as bioaerosols, dry chemicals and airborne particulates.

Surgical masks do not provide respiratory protection.

If you need an N95, full- or half-face N100, PAPR, or any other kind of fitted respirator, contact EH&S for a fit test.

Fit testing requires a medical evaluation.

See the LSUHSC Respiratory Protection Program for more information.

Gloves:

The type of glove (material) used is dependent upon the chemical being handled.

When using corrosive or toxic chemicals the gloves should be long enough to protect the forearm.

Long gloves can be cuffed at the bottom to help prevent chemicals from running down the arm.

Proper glove selection information can be accessed online at http://www.ansellpro.com/specware/

• Latex or nitrile gloves should be used for all handling of biological materials.
• Double gloves may be needed in some circumstances in order to avoid exposure or contamination.
• The type of gloves necessary and the frequency of changing is specific to your work and should be indicated in lab-specific training.
• Gloves must never be worn outside of the work area.

Goggles and Face Shields

Splash goggles must be worn whenever lab workers are using liquid chemicals that could injure the eyes, including heated liquids.

Face shields must be worn in conjunction with splash goggles if lab workers are using chemicals that could splash and corrode or burn the face, or when using toxic chemicals that could be splashed and absorbed through the skin.

PPE Storage

Personal protective equipment should be stored in a manner that protects the equipment from:

• Dust.
• Sunlight.
• Excess moisture.
• Extreme temperatures.
• Deformation.
• Chemical degradation.
• For more information see the PPE Policy.

Emergency Eyewashes and Showers Compressed Gasses and Cryogenics

Emergency Eyewashes and Showers

Eyewash and shower stations must be accessible to lab personnel that are using chemicals that could injure the eyes/skin.

If equipment or supplies are located too close to this safety equipment, you may not be able to freely access the shower or eyewash. Furthermore, the equipment could become damaged during use or testing. For more information see the Emergency Eyewash and Shower Policy.

Compressed Gas Cylinders

• Cylinders could act as a projectile if dropped and damaged.
• Secure cylinders to stationary objects (like a wall).
• Remove regulators and install valve protection caps when cylinders are not in use.
• If cylinder not properly secured, create an On-line Service request.

See the Compressed Gas Cylinder Policy for more information.

Liquid Nitrogen/Cryogenic

• (LN2)is extremely cold. -320°F (-196°C)
• Displaces oxygen and expands rapidly.
• Expands rapidly.
• Protect eyes and skin with goggles, gloves, shoes.

• Safe handling tips for (LN2)
• Do not allow to touch bare skin.
• Do not seal containers.
• Transfer liquid with care.
• Transport and handle containers with care.
• Handle containers with care.
• For more information see the Cryogenic Liquid Policy and the Liquid Nitrogen Supply and Safety Plan

More Information and Points of Contact

Further information on these and all LSUHSC Environmental Health and Safety programs can be found at the EH&S web site.

If you have questions on this training please contact Environmental Health and Safety at 568-6585 or safety@lsuhsc.edu.