Revised August 2019 CHEM 355 Lab Safety

PPE Personal Protective Equipment: What must be worn when you work in the laboratory. Eye Protection Lab Coat Long Pants Closed Toed Shoes – no exposed skin around feet Lab gloves – when required

Clothing and Foot Protection Clothing (with lab coat) must cover all exposed skin below the waist, including legs/ankles Stockings or leggings do not provide good coverage Sandals, flip-flops, Crocs, open-toe and open-top shoes and canvas shoes are not appropriate. These are not going to protect your feet if you drop a piece of glassware with a liquid chemical reagent in it.   

Eye Protection Contact lenses are okay as long as glasses/goggles are worn Prescription glasses – you must wear goggles over them Eye wash stations are present in all labs

Use of Gloves Remove gloves before handling objects such as doorknobs, phones, pens, or computer keyboards while in lab. It might be convenient to have one gloved hand and one ungloved hand to do procedures where these kinds of things are used. Throw away gloves any time you take them off. You should expect to use several pairs of gloves in any given lab period. Wear gloves when using the laptop attached to the Gel Doc, which is an ethidium bromide area.

Eyewash / Safety Shower The safety shower is on the right. Pull the handle and water will start spraying from the shower head on the ceiling. There’s no drain in the floor – we only do this in emergencies, because a flood of water will have to be cleaned up. The eyewash is on the left. Pull the handle and a fountain of water will appear that you can use to bathe your eyes.

Eye Wash

Safety Shower

Keep your lab area clean and free of hazards × × Don’t leave things on the floor because someone could trip. Don’t block the floor in front of the eyewash/shower station. × × Throw away used paper towels and used gloves, immediately.

Using the Fume Hoods Properly When using a laboratory hood, check that the airflow is in the normal range on the digital display Turn on the hood light Set the equipment and chemicals back at least 6 inches. Never lean in and/or put your head in the hood when you are working. It’s a good idea to put liquid reagent containers in trays to catch all spills and drips

Using the Fume Hoods Properly This window/bar is called the sash. If this is not saying NORMAL, then the hood is not protecting you. Keeping the sash and sliding panels in proper position keeps this NORMAL, otherwise the alarm goes off. If the alarm goes off, you need to reposition things to the correct positions, then press the “mute” button to reset the controller. The sash should never be raised above the green “operation” level when you are working in the hood.

✓ ✓ × ✓ In use, side-to-side panel used as shield Closed, not in use In use, sash (window) raised to less than 18 inches Don’t open side shields to make one big window. × ✓

Bunsen Burners When you light a Bunsen burner, make sure everyone working nearby you knows it is lit. (Also, every time you work at the bench in the lab that has burners, CHECK to see if they are lit as you first approach.) Do not leave a lit burner unattended. Bunsen burners must be turned off in TWO locations, any time they are not in use: Turn the valve at the bottom of the burner so that it is perpendicular to the stem of the burner. Turn off the gas valve on the bench.

Fire Alarms – know the location of one close to your lab

Fire Extinguishers – we have several in the labs and in the hallways.

Types of Fire Extinguishers This is a special fire extinguisher for combustible metal fires. It is a type D fire extinguisher. You won’t need to use this unless you work in a research lab with combustible metals. Most of our fire extinguishers are ABC. It contains a dry powder to put out the kinds of fires we might encounter in the chemistry labs where we have class.

Student Reaction in a Fire Although we want you to be informed on the operation of a fire extinguisher, we do not expect you to use it. If a fire is ignited in your area, the proper STUDENT response is to: Notify everyone in the room If possible shutdown any reaction in progress by removing heat/energy source Proceed to the nearest exit and pull the nearest fire alarm Evacuate the building Assemble in front of the library or in the YWCA parking lot for a positive headcount

Injury procedure First Aid kits are available in the lab with band aids and other items for treating small cuts and burns. Campus public safety can be reached at 3-5609 for non-emergencies. If it is a serious injury, call 911 for emergencies. The LiveSafe app can also be used to report emergencies and non-emergencies. Minor cuts or burns can be treated at the discretion of the instructor, but campus safety officials will be called if the instructor thinks it is necessary.

Centrifuge Safety

Rotor Safety Do not run rotors above their rated speed Inspect rotor for imperfections and signs of wear that can eventually lead to catastrophic failure Do not drop rotor Rinse large rotors after every use that might have spills Avoid using abrasive brushes for cleaning If you suspect rotor has been damaged, do not use it Do not use a rotor that is not compatible with your model centrifuge Use tubes and adapters that are rated for use in the rotor being used at or below the speed they are rated for Disposable tubes Fixed angle rotor Swinging Bucket rotor Need adaptors

Accident involving improper rotor usage Centrifuges that malfunction can create projectiles out of the rotor shards. If the centrifuge starts to make horrible noises, cut the power and leave the room

Loading the Centrifuge Only use tubes that you know are supposed to go into the rotor you are using at the speed you intend to use it Be certain that tubes are balanced with a partner Don’t forget to include caps when weighing the tubes for balance Secure the rotor on the spindle by tightening all knobs on the lid Tug gently on the rotor to make sure it is secured to the spindle Do not overfill bottles (most are 3/4 full max; all centrifuge bottles have specifications that you should look up before using them) Counterbalance your labeled sample Both knobs are tightened in some models to secure rotor to the spindle

Unloading Centrifuge Take precautions if biohazards or other hazardous material is used as aerosols can form during vacuum cycles Clean the chamber from condensation and any spills Never try to open the centrifuge door before the rotor is done spinning Never reach a hand or anything else into the chamber when rotor is spinning Note: it is sometimes difficult to look at a spinning rotor and determine if it is spinning

Centrifuge Safety http://www.youtube.com/watch?v=q_0phA034n0 -Do not walk away until the rotor has reached its running speed. -Note: A modern centrifuge will have low tolerance for mismatched tubes and will shut itself off if tubes are not balanced. -Also, most modern centrifuges will not allow the door to unlock while the rotor is still in motion. http://www.youtube.com/watch?v=q_0phA034n0

Autoclave Safety

What is an Autoclave? An autoclave is a specialized piece of equipment designed to deliver heat under pressure to a chamber, with the goal of decontaminating or sterilizing the contents of the chamber.

Personal Protective Equipment Autoclaves utilize steam, heat, and pressure; therefore, the risk of personal injury through scalding, burns and exploding glassware is great. Personal protective equipment is absolutely required. 1) Safety Glasses 2) Lab Coat 3) Long pants 4) Closed Shoes 5) Long thermal gloves 6) Face shield recommended

What can be autoclaved? Cultures and stocks of infectious material Culture dishes Tips, pipettes, gloves, paper towels, aluminum foil Centrifuge bottles Glassware -- all caps must be loosened (unless they are made of aluminum foil) Media and other aqueous solutions Solid items contaminated with cells

What CANNOT be autoclaved? Solvents or volatiles Chlorinated compounds (HCl, bleach) Corrosives Radioactive material Some plastics Note it would be very hazardous to everyone in the lab if these chemicals were autoclaved! Therefore, it is very important that you only put autoclavable items into the Biohazard Trash to Be Autoclaved containers.

Cycle Differences Fluids must be autoclaved under a “liquid” setting Items such as pipette tips, test tubes, and centrifuge bottles are run under “dry” or “gravity” setting The difference in settings is how the cycle is vented Liquids must depressurize slowly and dry cycles conclude with a vacuum step to draw off condensation

Loading and Unloading the Autoclave All screw caps must be loosened to prevent pressure changes in the glassware that can cause the container to burst All items should be placed in an autoclave tray to prevent scald burns in the event of a spill Return autoclave trays promptly so that other users do not skip using a tray because they can’t find one Don’t skip using a tray Do not remove liquid that is still boiling If possible, allow glassware to cool before removing Loosen cap by several threads

Autoclave Door Safety Never try to open a door that is under pressure Never try to speed up the venting process by tampering with the door, by turning on and off the machine, etc. Venting takes time. Know where the pressure gauges are for the instrument you are using If possible, vent door slowly

Autoclaving Waste This bag is too full Contaminated pipette tips and solid waste should be sterilized prior to disposal Collect waste in a special autoclave-safe biohazards bag Place bag in secondary container Vent the bag by opening Do not overfill bag After removal place entire bag in a new trash bag so that “biohazard” signs are no longer showing Sterilized waste can go into the normal trash Autoclave tape can be used to verify heat delivery but it does not guarantee proper sterilization

Autoclave Container Choice Pyrex glass, metal, polypropylene (PP) plastic and polycarbonate (PC) plastic are best choices Polyethylene (PE), polystyrene (PS), and high density polyethylene (HDPE) will often melt and make a mess

Autoclaving Tips for Liquid Loads Add a 2-cm depth of water to trays with glassware; the water helps eliminate air pockets between the tray and the glass and helps prevent glass from breaking Do not fill liquid containers past 75% volume Separate items to increase steam penetration Increase cycle time for large volumes of liquid Liq7 = for containers with < 500 ml Liq8 = for containers with > 500 ml Temperature must be maintained at 121°C for at least 30 minutes for liquid loads

Autoclaving Tips for Dry Loads Do not add water to the tray Our autoclave dry load setting is called either Grav30 or PreVac1

Autoclave Use and Maintenance If the autoclave is asleep when you walk up to it, you must log in then wait 30 minutes before running your cycle This time is necessary for the steam generator to warm up. Running a cycle before this time will result in incomplete sterilization and potential damage to the steam generator. Only use the autoclave if it is not currently running a cycle and no one is signed up on the autoclave calendar If you know ahead of time that you will be using the autoclave, sign up on the calendar in advance. Liq7 and Liq8 cycles are both somewhat shorter than 2 hours. Report any irregularities Record any errors in the logbook then immediately email Dr. Stephanie Boussert

Other Biochemistry Safety Concerns

Toxic and Health Hazardous Chemicals Categories: Irritants Sensitizers Corrosives Carcinogens Target Organ Effects Reproductive Health Toxins Acute Toxins Physical Health Hazards Common routes of exposure in the lab are inhalation and skin absorption, while ingestion is less common. Carcinogens, reproductive toxins, target organ damage Acute toxicity, fatality Source: Basic & Clinical Pharmacology, 12e >Chapter 56. Introduction to Toxicology: Occupational & Environmental Daniel T. Teitelbaum, MD* Categories list is from Harvard Lab Safety Manual GHS labels Irritants, sensitizers, acutely toxic Corrosives

GHS Symbols

Toxicity and Minimizing Exposure Toxic reactions depend on the duration of exposure Acute exposure – a single exposure, or multiple exposures over 1-2 days Chronic exposure – multiple exposures over a longer period of time The Department of Labor OSHA establishes legal permissible exposure limits (PELs) for the workplace “However, these standards must not be taken to represent an absolute boundary between the positively safe and the positively unsafe.” – OSHA website LD50 is a measure of acute toxicity LD50 is the lethal dose of a chemical required to kill 50% of a test animal population (measured in mg chemical per kg body weight). The lower a chemical’s LD50, the more toxic it is.

Carcinogens Genotoxic carcinogens cause DNA damage directly (e.g., by forming a DNA adduct) and/or indirectly (e.g., by producing reactive oxygen species that inflict genomic damage). If unrepaired before replication, DNA damage results in a mutation. Mutations can result in tumor initiation if they occur in genes related to cell division, programmed cell death, DNA repair, etc. Non-genotoxic carcinogens promote carcinogenesis without damaging DNA For example, these chemicals might stimulate cell proliferation, tissue invasion, or angiogenesis by binding to a receptor. These carcinogens mostly cause tumor promotion. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12e > Section IX. Special Systems Pharmacology > Chapter 67. Environmental Toxicology: Carcinogens and Heavy Metals Michael C. Byrns and Trevor M. Penning

Toxic Chemicals In Biochemistry When working with toxic chemicals, extra attention should be paid to selection and use of PPE. Protect yourself by using PPE properly and disposing of contaminated PPE. Protect others by not spreading the toxic chemical around the lab. Many chemicals commonly used in biochemistry are toxic. A few common examples are detailed on the following slides.

Ethidium Bromide Ethidium bromide is an intercalating agent commonly used as a fluorescent label in molecular biology laboratories for techniques such as agarose gel electrophoresis. Avoid working with the powder, which can be fatal when inhaled (instead work with solutions). Handle in hood Mark areas of use and decontaminate frequently Do not heat agarose with ethidium bromide in it Use gloves; absorbs through skin

Acrylamide Acrylamide is the monomeric precursor to polyacrylamide used in SDS-PAGE. May cause cancer. May cause heritable genetic damage. Also toxic in contact with skin and if swallowed. Danger of serious damage to health by prolonged exposure through inhalation, in contact with skin or if swallowed. Avoid working with the powder due to inhalation hazard (instead work with solutions) Use gloves when handling Polymerize excess solution for safer disposal

Sodium Azide Sodium azide is commonly found in dilute solutions to prevent bacterial growth. The acute toxicity of sodium azide is high Do not allow sodium azide to come into contact with heavy metals or their salts, because a reaction may form heavy metal azides, which are explosives. Do not allow sodium azide to come into contact with aqueous acids, because reaction liberates highly toxic hydrazoic acid, which is a dangerous explosive.  Containers should be stored in secondary containers in a cool, dry secured storage area separated from acids.  Avoid using metal spatulas  Do not dispose of solutions down the drain, as explosions could result.

Phenylmethanesulfonyl Fluoride PMSF is used in solution to inhibit proteases Toxic if swallowed. Causes severe skin burns and eye damage. Extremely destructive to tissues of mucous membranes and respiratory tract. Corrosive. Target Organs: Nerves, Heart, Blood, Eyes. Wear gloves. Take extra precautions when working with the powder. Do not leave any traces of spilled power on the bench, balance, etc., where it could endanger another lab user.

Toxic Chemicals in Other Branches of Chemistry Many chemicals commonly used in other branches of chemistry (e.g., synthetic chemistry) are also toxic. Some examples: Halogenated aliphatic hydrocarbons (e.g., chloroform, carbon tetrachloride, etc.) – cause central nervous system depression, liver injury, kidney injury, and some degree of cardiotoxicity. Many are carcinogenic. Aromatics (e.g., benzene, toluene, xylene) – cause central nervous system depression, skin irritation. Benzene causes bone marrow injury and is associated with leukemia. Source: Basic & Clinical Pharmacology, 12e >Chapter 56. Introduction to Toxicology: Occupational & Environmental Daniel T. Teitelbaum, MD*

Toxic Chemicals and Material Safety Data Sheets (MSDS) Identify toxicity hazards for any chemical by consulting the MSDS. The Hazards section includes toxicity warnings:

Toxic Chemicals and Material Safety Data Sheets (MSDS) Look for the health NFPA category rated from 0-4, and read warning statements. 0 – Hazard no greater than ordinary material 1 – May cause irritation; minimal residual injury 2 – Intense or prolonged exposure may cause incapacitation; residual injury may occur if not treated 3 – Exposure could cause serious injury even if treated 4 – Exposure may cause death

Toxic Chemicals and Material Safety Data Sheets (MSDS) Separate sections indicate toxicological data on the chemical:

Biological Hazards Biological hazards are potential sources of infectious agents that could be harmful to human health. Bacterial, fungal, parasitic, viral, and prion agents. Sources may include animals, tissues, cells, blood, and nucleic acid samples, including recombinant DNA. Biohazard safety level 1 – microbes are not known to consistently cause disease in healthy adults and present minimal potential hazard to lab workers and the environment. An example of a microbe that is typically worked with at a BSL-1 is a nonpathogenic strain of E. coli. Work requires our usual PPE and hand-washing procedures as well as decontamination procedures (all plastic materials in contact with the cells are autoclaved; all glassware and liquid waste is treated with bleach)

High Voltage Techniques DNA and protein gel electrophoresis combine high voltage with the use of aqueous solutions Take precautions to avoid electrocution. Modern gel boxes have electrodes positioned on the lids to drastically reduce the risk of electrocution. Always secure the gel box lid before turning on the voltage. Turn off the voltage before removing the lid to a gel box. Match the red and black electrodes to the corresponding red and black outlets on the power supply.

Ultracold (-80°C) Freezer Use Many biological samples and chemicals need to be preserved at temperatures below room temperature Always consult the label: 4°C (refrigerator), -20°C (conventional freezer), -80°C (ultracold freezer), -196°C (liquid nitrogen) Cold storage can slow cell death rate, preserve enzyme activity, inhibit contaminating bacterial growth, and prevent degradation. Use insulated gloves to handle ultracold materials. Handle glass dewars with caution – danger of exploding glass if they are knocked over and broken. Keep the -80° freezer closed!

Liquid Nitrogen Safety Liquid nitrogen (LN2) is commonly used to rapidly freeze proteins and bacteria LN2 rapidly evaporates and can displace air in enclosed spaces causing suffocation LN2 can cause death of human tissue from extreme cold Minor contact can cause “burns” Evacuated glass dewars can sometimes burst unexpectedly LN2 can condense liquid oxygen

Liquid Nitrogen DON’T’s DON’T use in confined space DON’T freeze items in centrifuge tubes with snap caps DON’T transport LN2 in a closed automobile DON’T transport LN2 in a passenger elevator DON’T allow a storage dewar to tip over DON’T leave cold fingers on a vacuum line in LN2 overnight DON’T use without PPE! NO!

Liquid Nitrogen Do’s YES! YES! DO use or dispense LN2 only in well ventilated areas DO ensure glass dewars are taped or wrapped DO use approved containers only such as a dewar or threaded cryovials for storage DO make sure any vessel with LN2 is VENTED DO secure storage dewars against spilling DO use appropriate PPE which includes: Face shield (or minimally goggles) Long thermal gloves Apron or lab coat Closed toed Shoes Long pants NO! YES!

Report any concerns If you have any safety concerns about the lab you are working in or the people working around you, you can contact: Your lab instructor Dr. Marcello Forconi – Head of the departmental safety committee Dr. Pamela Riggs-Gelasco – Department Chair for Chemistry and Biochemistry Dr. Jim Deavor, Associate Dean of the School of Science and Mathematics.