Laboratory Conduct & Biosafety

Introduction

Working in a research laboratory requires being aware of the different types of hazards we use or have in the lab, how to handle them safely, and what to do in the event of a spill or an exposure incident. For the most part, we follow the Johns Hopkins Safety policies, and much of this material is covered in your required safety trainings. Additional biosafety information is available in the Biosafety in Microbial and Biomedical Laboratories (BMBL) handbook, currently on its 6th edition. We function at BSL-1 and BSL-2 levels, so we encourage you to review these chapters in the BMBL.

We do have a few unique standard operating procedures (SOPs) that describe how we work in the lab. These are detailed in the following sections and will be taught to you by Mike and/or your primary laboratory mentor. The most important thing here is to ask your mentor, ask Mike, or ask the Biosafety office if you have ANY questions. We want to all stay safe and keep the lab a secure and fun place to work, so IF YOU ARE UNSURE, ASK SOMEONE.

Safety Training

Stay up to date with your required safety training. Online training courses can be found on the Hopkins Medicine HSE training page. Please take each of the following training courses, and any additional courses relevant to your project or that you think may help you operate safely.

Required of each Piacentino Lab member:

  • Laboratory Safety/Biosafety Levels I & II (virtual, instructor-led)
  • Bloodborne Pathogens Orientation (online, user-driven)
  • Fire, Hazard Communication and Construction Safety (online, user-driven)
  • Chemical Waste Management (online, user-driven)

After completing these trainings, keep an eye out for notifications in your email for training refreshers. Safety procedures change as we develop new ways to mitigate risk, so it is important to stay up to date and renew our training periodically. Some of these courses require re-training every year or two.

Safe Laboratory Conduct

Some general rules in lab:

  • Always work in a safe manner and with a professional attitude
  • No food or drink, or storage of food/drink for human consumption is allowed in lab
  • No animals or plants are allowed in the laboratory unless they are part of the work being performed or approved by Mike and the Biosafety office
  • No handling cosmetics, contact lenses, earrings, or other personal items that may introduce hazardous materials into the body/face
  • Wash your hands after performing any and all laboratory procedures
  • Wear appropriate clothing and PPE in the laboratory and while performing work
  • Keep our lab clean, tidy, and disinfected
    • Keep an organized bench with clearly labeled solutions
    • Tidy any common space you use so it is safe for the next user
    • Wipe down/decontaminate all workspaces following experiments, and always decontaminate the area following a spill
  • All incidents which result in an injury to any lab members or visitors must be appropriately documented and reported – contact Mike immediately following any incident

Laboratory Attire & PPE

The purpose of appropriate laboratory attire and PPE is to protect our skin and bodies from exposure to hazardous materials.

Shorts, miniskirts, or any apparel that does not cover the skin above the knee when seated shall NOT be worn in the laboratory without appropriate overprotection (a buttoned lab coat that extends past the knee). Open-toed shoes, sandals, or shoes made of loosely woven materials are not permitted when working in the lab.

PPE are an additional layer of protection that should always be worn as appropriate to the laboratory task. Most of our work falls under Biosafety Level 1 (BSL-1) classification and should be performed while wearing disposable gloves.

Our tissue culture work (in WBSB 104A) and working with chicken eggs that have not been confirmed Salmonella-clean fall under BSL-2 classification. When working in these systems, wear disposable gloves, a lab coat, and safety goggles when producing aerosols.

When working with cryogenic hazards (liquid nitrogen, dry ice), additional PPE is required including cryogenic gloves, impermeable shoes and apron, goggles, and a face shield.

Dispose of your gloves after use, and never touch your computer, doorknobs, or common areas while wearing gloves. Always remove your gloves before leaving the lab, or remove one glove to handle doors if transporting materials in the other gloved hand.

Hazards in the Lab

This lists the most significant hazards we keep in the Piacentino lab. If you notice something not on this list, let Mike know, and we’ll add it! Before using any of these items, please seek appropriate training from Mike and/or your designated lab mentor.

Physical Hazards

  • Sharp hazards: glass (including broken beakers, flasks, slides, and coverslips), needles (glass microinjection needles and syringe needles), dissecting tools (forceps, scissors, and knives), razor blades
  • Electrical hazards: all major lab equipment must be connected directly to a power outlet, no power strip; all power strips must be elevated >12 inches off the floor in case of flooding; do not daisy-chain electrical cables; cover any cables that cross a walk-space, otherwise it’s a tripping hazard
  • Compressed gas cylinders: these can become a rocket if they fall over and/or have a broken regulator; always keep compressed gas strapped to a secured surface using chains or straps rated for this purpose; always use a cart to transport gas cylinders
  • Cryogenic hazards (liquid nitrogen and dry ice): appropriate PPE (especially for liquid nitrogen) includes covering all skin, cryogenic gloves, impermeable shoes and apron, goggles and face shield. Liquid nitrogen and dry ice can pose an asphyxiation hazard since they can displace oxygen rapidly – work with cryogenics in open spaces with good ventilation, NEVER use or put these items in a cold room, and NEVER seal cryogenics in an air-tight container, as it will become explosive
  • Laser hazards: many microscopes use laser lines to excite fluorescent samples; misuse of lasers can result in physical damage, can start a fire, and can result in blindness

Chemical Hazards

Safety Data Sheets (SDS) are found in the lab safety binder, and can be accessed online. Below are the chemical hazards we house and use in the laboratory, and their respective hazards:

  • Fixatives: paraformaldehyde (flammable, corrosive, irritant, acute toxicity, chronic health hazard); glutaraldehyde (corrosive, chronic health hazard, acute toxicity, aquatic toxicant)
  • Acids and bases: hydrochloric acid (corrosive); acetic acid (corrosive, flammable); sodium hydroxide (corrosive)
  • Alcohols: ethanol (flammable, irritant, acute toxicity); isopropanol (flammable, irritant, acute toxicity); methanol (flammable, acute toxicity, chronic health hazard)
  • Other: phenol (corrosive, acute toxicity, chronic health hazard); chloroform (acute toxicity, chronic health hazard); ethidium bromide (acute toxicity, chronic health hazard, carcinogen)

Biological Hazards

  • Biosafety Level 1 (BSL-1) hazards: E. coli K12 (DH5α) strains; recombinant DNA molecules handled in or extracted from E. coli that cannot infect or integrate into cells; chicken eggs that are verified as Salmonella-clean (supplied by UConn Poultry)
  • Biosafety Level 2 (BSL-2) hazards: human-derived or non-human primate-derived cell lines (including U-2 OS cells); any recombinant DNA molecules used in tissue culture experiments; chicken eggs that are UNverified as Salmonella-clean

Biohazard SOPs

This section details the primary biohazards we use in the lab, how to handle and dispose of them safely, and how to clean up spills or messes with these agents. We have IBC approval for these procedures, and documentation of our risks and handling procedures. Please read the material below and also look at our registered protocols that are printed in our lab safety binder.

E. coli SOP (BSL-1)

We perform routine molecular biology work using E. coli bacterial strains (like DH5α) to amplify our plasmid DNAs. These experiments can be performed safely at your laboratory bench following standard BSL-1 practices. Importantly:

PPE: When working with bacterial cultures, wear gloves at all times. If performing a procedure that produces aerosols, wear safety glasses to protect from splashes into your eyes. When finished working, dispose of your gloves and any contaminated materials in the red-bagged biohazard box and wash your hands.

Disposal: Any bacterial waste must be disposed of as biohazardous materials.

  • Solids (including LB plates, gloves, pipet tips, serological pipets, disposable culture tubes, etc) are disposed of in red-bagged biohazard boxes for subsequent pickup and incineration. Before tossing your LB plates, please wrap them in parafilm to contain the bacteria.
  • Liquids produced during bacterial work (including bacterial cultures, LB and SOC broths, etc), are inactivated by incubation with bleach (10% final concentration) for 30 minutes. After inactivation, liquid waste can be rinsed down the sink with excess water.

Cleanup and disinfection: After working with bacterial cultures, always disinfect your workspace (surfaces, tools, etc) with 70% ethanol and paper towels. In the event of a large spill, follow the Spill Clean Up protocol below. When finished cleaning, dispose of your gloves, and wash your hands.

Chicken Egg SOP (BSL-2)

We frequently receive fresh fertilized chicken eggs which will be incubated to desired embryonic stages in the lab, manipulated at the designated embryology spaces, then cultured either ex ovo or in ovo. You will get hands-on training as is appropriate for your research project. Here we detail the primary safety concerns.

The primary concern with chicken egg material is the potential for Salmonella transmission, which can pose a human health risk if ingested; Salmonella is designated as a BSL-2 biohazard. We receive fertilized eggs from distributors that are part of the National Poultry Improvement Plan (NPIP) – a program committed to diminishing the spread of Pullorum Disease. Under this program, these distributors maintain farming practices free of Salmonella, as well as Pullorum Typhoid and Avian influenza. Certification is provided with each shipment and will be verified upon receipt.

PPE: When handling egg materials, always wear gloves and a lab coat. If producing aerosols or at risk of splashing, also wear eye protection. A lab coat is optional but encouraged if you can confirm the eggs are Salmonella-clean. We don’t want to spill eggs on our clothing! To protect your workspace and make cleanup easy, always cover your working area with absorbent bench paper. When finished working, dispose of your gloves and any contaminated materials in the red-bagged biohazard box and wash your hands.

Disposal: Experiments with unfixed egg/embryo material will be performed over benchtops covered with disposable bench paper to absorb any spills, and the bench paper will be collected into red-bagged biohazard boxes. At the end of each experiment, all bagged egg waste will be frozen at -20°C and stored in the freezer in WBSB 104 until disposal. When 3-4 experiments worth of frozen egg waste are accumulated, we will transfer these bags into a fresh, red-bagged biohazard box which will be removed for incineration through standard biohazard waste streams.

Cleanup and disinfection: Clean up after each experiment by collecting all disposable materials into your egg waste bag. Wipe down the work area with 70% ethanol and paper towels to prepare the workspace for the next user. Small spills will be cleaned with paper towels and water first to pick up as much egg material as possible. Then disinfect the area with 70% ethanol (ethanol will fix liquid egg material to the bench if not wiped up with water first) and paper towels. Contaminated materials will be bagged, and frozen with the remaining experiment material. In the event of a large spill, follow the Spill Clean Up protocol below. When finished cleaning, dispose of your gloves, and wash your hands.

Working with Fixatives SOP

Preparing stocks and fixing tissues

We frequently fix embryonic tissue with hazardous fixatives such as paraformaldehyde and glutaraldehyde. Spills and inhalation during use can pose serious risks to your health, and appropriate measures are required to mitigate these risks.

When to follow this: Always work in the chemical fume hood when…

  1. Making 4% Paraformaldehyde (PFA) stocks, as heating solutions to promote dissolving of PFA powder releases strong hazardous fumes
  2. Fixing embryos/tissue – bring your live tissue to the fume hood and pour PFA into your desired dishes. Add your embryos to the liquid in the hood and leave to fix. Return to the hood for the first wash after fixation (collecting fixative and the first wash into appropriately labeled bottles), then you can remove your fixed tissue to return to the embryology station for dissection.

PPE: When handling hazardous chemicals, always work in a chemical fume hood (one in WBSB 104 and another in WBSB 108), and wear gloves and a lab coat. The hood is designed to ventilate hazardous fumes so that you do not inhale them. This is especially important when fixatives are heated (e.g. during preparation of 4% paraformaldehyde (PFA) stocks). Discard your gloves after working with fixatives.

Disposal: See Lab Waste Handling & Disposal, Hazardous Liquids (Chemical). Notably, fixatives cannot be poured down the drain and must be collected in labeled containers stored in our Satellite Accumulation Area (SAA) within the fume hood. After fixing tissue, discard fixative waste and the first “non-hazardous” wash into the appropriately labeled waste bottle.

Cleanup and disinfection: Clean up after each experiment by removing all your materials from the fume hood and leaving the space open and clear for the next user. Wipe down the work area with 70% ethanol and paper towels. When finished cleaning, dispose of your gloves, and wash your hands.

Tissue Culture SOP (BSL-2)

We grow cells in tissue culture in WBSB room 104A. These cell lines include chicken-derived tissues (like DF-1 fibroblasts), but also some cell lines from human-derived samples (like U-2 OS osteosarcoma cells). Whenever working with human-derived materials there is an elevated concern for possible exposure to bloodborne pathogens. As such, we treat all active tissue culture material as a BSL-2 biohazard. Mike or your lab mentor will train you in safe tissue culture procedures before you work alone with these materials.

PPE: When working in WBSB 104A on any tissue culture experiment, wear gloves and a lab coat. Handle all materials within the Biosafety cabinets (BSC) with the airflow vents activated. If operating outside of the BSC, wear safety goggles to protect against splashes. When finished working, dispose of your gloves and any contaminated materials in the red-bagged biohazard box and wash your hands.

Disposal: Any tissue culture waste must be disposed of as biohazardous materials.

  • Solids (including plates, gloves, pipet tips, serological pipets, tubes, etc) are disposed of in red-bagged biohazard boxes for subsequent pickup and incineration.
  • Liquids produced during tissue culture work (including media and buffers, resuspended cells, etc), are inactivated by aspiration into a vacuum flask and incubation with bleach (10% final concentration) for 30 minutes. After inactivation, liquid waste can be rinsed down the sink with excess water.
  • We frequently perform immunohistochemistry for visualization of cells from tissue culture experiments. For these experiments, fix cells in the BSC with an appropriate volume of 4% paraformaldehyde (generally 200-1000 µL) for 10 minutes at room temperature. Fixed cells are effectively inactivated and can be taken out of the BSC for subsequent labeling and imaging.

Cleanup and disinfection: After completing your work in the BSC, remove all materials and disinfect the surfaces and tools with 70% ethanol and paper towels. Close the BSC sash, turn off the airline, and turn on the UV lamp for 30 minutes to disinfect the cabinet. Bleach and dispose of all liquid waste as described above, remove your PPE and dispose of gloves in the red-bagged biohazard bins. Wash your hands before leaving the tissue culture area.

Lab Waste Handling & Disposal

It is our responsibility to protect not only ourselves and our teammates but the Johns Hopkins community and beyond. This means being aware of how we dispose of materials in the lab to prevent chemical hazards or biohazards from leaving our controlled environment and posing a risk to others and the environment. While we know what we are using and their inherent risks, it is essential that we take waste disposal seriously and treat all waste produced in our lab as a potential chemical or biohazard risk. Below we detail how to handle the major classes of waste you will encounter and how to safely dispose of these materials. If you’re unsure, what should you do? ASK SOMEONE!

Solids: Almost all solids we produce in the lab must go into the red-bagged biohazard boxes. These boxes are taken for incineration to ensure no release of biohazardous materials. All gloves, soiled paper towels, rigid plastics (tubes, plates, tips, serological pipets, etc), chicken eggs and embryos, tissue culture supplies, ethidium bromide-stained gels, and even autoclaved solids must be disposed in this box. Never remove the red bag liner from the box.

When the box is 70% full:

  • Tape the red bag shut
  • Cover the box with the included cardboard lid and seal it with tape
  • Label the top with “Piacentino Lab, WBSB 104” and the date
  • Place the box in the hallway for collection
  • Immediately set up a new red-bagged biohazard box

Uncontaminated paper/plastic products and sharps (needles, razor blades) are the exception and do not go in the red-bagged biohazard box. Uncontaminated paper/plastic products (catalogs, journals, notebook paper, cardboard boxes, and empty pipet tip boxes) can be disposed in clearly labeled recycling containers. If you’re not sure if it became contaminated, treat it as if it is a hazard and put it in the red-bagged biohazard box.

Sharps: Sharps waste have their own dedicated container – smaller, red plastic sharps containers with appropriate biohazard labels. Any sharps to be disposed (needles, razor blades, etc.) should be placed in a plastic sharps disposal container. NEVER reach into a sharps container.

When the sharps container is filled to the indicated line:

  • Close the lid of the sharps container
  • Label with “Piacentino Lab, WBSB 104” and the date
  • Place the box in the hallway for collection
  • Immediately set up a new sharps container

Non-Hazardous Liquids: We will produce a variety of liquid wastes, the majority of which are non-hazardous. These include water, buffer solutions used in immunostaining, in gel electrophoresis, etc. These solutions can be disposed of down the drain with plenty of water.

Hazardous Liquids (Chemical): Several solutions we use routinely in lab contain hazardous chemicals that CANNOT be poured down the drain. These include fixatives (paraformaldehyde, glutaraldehyde), methanol, chloroform, phenol, etc. When working with these hazardous liquids (e.g. fixing embryos, performing phenol/chloroform extractions, etc), work in the chemical fume hood until the samples have been sufficiently washed out of hazardous chemicals and collect used liquid in our Satellite Accumulation Area (SAA) in the chemical hood in WBSB 104. Add your solution to the appropriate waste container of matching waste. DO NOT MIX chemical waste. Keep waste containers in a secondary container (tub underneath them), and make sure they are clearly labeled with the contents. We will dispose of filled chemical waste bottles during the next chemical waste collection on the ground floor of PCTB (room G06 from 1-2pm on Wednesdays). Before bringing down for drop-off, fill out and print the chemical waste form.

Hazardous Liquids (Biohazard): We work with a few liquids that classify as a biohazard: 1) chicken egg waste, 2) media from bacterial work, and 3) liquids from tissue culture work. Chicken egg waste should be frozen at -20°C and disposed of in a fresh red-bagged biohazard box; do not package with any materials that can puncture, like pipet tips. Liquids produced from bacterial or tissue culture work must be inactivated before disposal. To inactivate these liquids, add bleach to a 10% final concentration and incubate for at least 30 minutes. After this incubation, the biohazards are effectively inactivated, and this waste is now safe to rinse down the sink with plenty of water.

If you are EVER unsure of how to dispose of something, ask Mike, ask a more senior lab member, or ask the Biosafety office.

Sharps Handling

We use several sharps in the lab routinely, including needles (metal and glass), glass slides, coverslips and bottles, razor blades, and dissecting tools. Sharps can pose a threat because they can puncture or cut our skin. While this is unpleasant, it also can introduce biohazards into our bodies or make us more susceptible to future exposures. Here are some tips for using sharps safely:

  • Minimize the handling of all sharps
  • Never recap needles or use any technique that involves pointing a needle or sharp toward the body (yours or another’s)
  • Do not remove a used needle from a syringe or a blade from a handle – dispose of the whole item in the sharps bin
  • Shearing, breaking, or bending of needles is prohibited
  • Promptly dispose of your sharps in the appropriate containers
  • NEVER jam sharps into a container
  • NEVER insert your hand or fingers into a sharps container – if it is necessary to open and retrieve something from a sharps container, call HSE at x5-5918
  • Use a utensil such as tongs, forceps, or dustpan and broom to pick up any broken glass, needles, or sharps that have fallen on the floor or another surface

Needlesticks & Exposure

All needlesticks and exposures to sharps must be reported immediately by calling the Needlestick Hotline 5-STIX (955-7849) for the East Baltimore Campus. An incident report must be filed. Specific details concerning the type of sharp and the actual occurrence should be listed to identify practices or types of equipment which may need to be modified.

Problems involving a particular type of sharp or procedure that may cause exposure to blood-borne pathogens should be brought to the attention of Health, Safety and Environment (5-5918).

Spill Clean Up

Spills happen, and usually, we can take care of them quickly and safely. When working with chemicals or biohazards, we need to take extra precautions to decontaminate the spill area before returning to work.

Take this time to identify where our lab spill kit is located: PPE (gloves, lab coats, goggles/face shield), broom and dust pan, absorbent pads, autoclave bags, red-bagged biohazard boxes, bleach or other disinfectant and container for mixing.

For Biological Spills

Small (<50 mL)

  1. Remove contaminated clothing and PPE and place in biohazard box or set aside for decontamination.
  2. Replace any removed PPE and put on a lab coat, double gloves, and eye protection.
  3. Cover spill with absorbent materials saturated with fresh decontamination solution (10% bleach). Spread the bleach around the outside of the spill and work inward, covering an area that extends greater than the visible spill area.
  4. Allow decontamination to sit for 30 minutes, then collect these materials.
  5. Dispose of all cleanup materials in the biohazard box.
  6. Remove lab coat, eye protection, and gloves; wash hands thoroughly.
  7. Notify Mike.

Large (>50 mL)

  1. Alert the room and isolate the area from traffic. If potentially hazardous aerosols have been generated, leave the area immediately and let the aerosols settle and building exhaust clear the area for 20-30 minutes. Otherwise, without risking exposure, surround the spill with absorbent material to contain spreading.
  2. Remove contaminated clothing and PPE and place in biohazard box or set aside for decontamination.
  3. If you can clean the spill without help, replace any removed PPE and put on a lab coat, double gloves, and eye protection.
  4. Cover the perimeter of the spill and then work inward to cover the spill itself with a decontamination solution (10% bleach) – cover the area with absorbent materials, larger than the actual spill to catch any unseen splatter.
  5. Wait 20-30 minutes, then dispose of materials in a biohazard box.
  6. Remove lab coat, eye protection, and gloves, and wash hands thoroughly.
  7. Notify Mike.

For Chemical Spills

  1. EVALUATE the spill: are the materials innocuous, corrosive, flammable, toxic or explosive? Identify all materials by common or chemical name, estimate how much is spilled, and evaluate the degree of danger to yourself, the lab, and equipment/property.
  2. CONTAIN the spill: utilize any action to prevent the spilled material from spreading and causing increased damage.
  3. EVACUATE the area if the spill cannot be contained, or if the spilled material produces irritating odors, flammable or explosive vapors.
  4. COMMUNICATE to the rest of the lab that there is a spill, and notify Mike.
  5. CLEAN up the spilled materials:
    • Innocuous materials can be cleaned up by us in the lab using paper towels or absorbent pads.
    • Spills of acids, bases or flammables and mercury can be cleaned up by lab personnel with proper PPE and neutralizers/absorbents.
    • Spills of toxic or explosive materials, or large spills of corrosive or flammable materials, should be handled by HSE – call the Haz-Techs at x5-4444 and have this information ready: your name and phone number, precise location of spill, exact description of what spilled (state any compounds that may form toxic compounds), any steps you have taken to control the spill, and any injuries that have occurred.
  6. DISPOSE of all contaminated materials.
  7. Consult HSE at x5-5918 with any questions regarding chemical or biological spills and spill cleanup.

Emergency contact numbers (fire, medical emergency, security, biological/chemical exposure and spill lines) are posted physically throughout the lab – familiarize yourself with their locations as part of your onboarding.