Biochemistry Lab Safety 1 PPE Personal Protective Equipment

Biochemistry Lab Safety

1. 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 2

Eye Protection • Contact lenses are OK as long as glasses/goggles are worn • Prescription glasses – you must wear goggles over them • Safety goggles are provided in organic labs in UV irradiating cabinets • Eye wash stations are present in all labs 3

Clothing and Foot Protection • Clothing must cover all exposed skin including legs/ankles • Stockings or leggings do not provide good coverage • Sandals, flip-flops, Crocs, open-toe and open-top (i. e. ballet flat) shoes and canvas shoes (i. e. Toms) are not appropriate. These are not going to protect your feet if you drop a piece of glass with a liquid chemical reagent in it. 4

Use of Gloves Remove gloves before handling objects such as doorknobs, telephones, pens, computer keyboards, p. H meter or other electronic buttons, or phones while in lab. It might be convenient to have one gloved hand one ungloved hand to do procedures where these kinds of things are used. • Throw away gloves anytime you take them off. • You should expect to use several pairs of gloves in any given lab period. • Glove video 5

Eyewash / Safety Shower The eyewash is on the left. Pull the handle and a fountain of water will appear that you can use to bathe your eyes. 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. 6

Eye Wash 7

Safety Shower 8

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 9 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. × 10

• 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. This is worse than doing the experiment with no hood at all. • It’s a good idea to put liquid reagent containers in trays to catch all spills and drips 11

Fire Alarms – know the location of one close to your lab 12

Fire Extinguishers – we have several in the labs and in the hallways. 13

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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. 15

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: 1) Notify everyone in the room 2) If possible shutdown any reaction in progress by removing heat/energy source 3) Proceed to the nearest exit and pull the nearest fire alarm 4) Evacuate the building 5) Assemble in front of the library or in the YWCA parking lot for a positive headcount 16

Keep your lab area clean. × Throw away used paper towels and used gloves, immediately. × Don’t block the floor in front of the eyewash/shower station. × Don’t leave cords dangling because someone will trip over them. × Don’t leave things in the floor because someone will trip over it. 17

Once again, the number to call in an emergency is: 843 -953 -5611 Please take a moment now to program this number into your cell phone. 18

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 the rotor after every use • 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 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 • 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 (3/4 full max) 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

Safety Overview • http: //www. youtube. com/watch? v=q_0 ph. A 0 34 n 0 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.

Autoclave Safety 25

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 and 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 Media and other aqueous solutions Contaminated solid items

What CANNOT be autoclaved? • • • Solvents or volatiles Chlorinated compounds (HCL, bleach) Corrosives Radioactive material Some plastics

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

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

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 • 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 past 75% volume • Separate items to increase steam penetration • Increase cycle time for large volumes of liquid • Temperature must be maintained at 121°C for at least 30 minutes

Maintenance • Report any irregularities to your supervisor • Do not operate if there is a steam outage • Failed runs should be reported and logged

Overview • http: //www. youtube. com/watch? v=T 901 F 2 W 7 wks • Please note: newer autoclaves such as the one in the New Science Center do not have pressure gauges and a chart recorder, but these parameters are displayed on the computer screen as the cycle is started.

Toxic Chemical Safety

Health Hazardous Chemicals • Categories: – – – – Irritants Carcinogens, Acute toxicity, fatality Sensitizers reproductive toxins, target organ damage Corrosives Carcinogens Target Organ Effects Reproductive Health Toxins Irritants, sensitizers, Corrosives acutely toxic Acute Toxins Physical Health Hazards • Common routes of exposure in the lab are inhalation and skin absorption, while ingestion is less common.

Toxic Chemicals • Many chemicals commonly used in the lab are toxic. – Toxicology concerns the degree to which a chemical is hazardous to human health. • How do toxicologists predict which chemicals will be toxic and determine their mechanism of action? – Animal studies to determine a dose-response curve to predict a threshold level above which a chemical is toxic – Mechanistic studies to determine how a chemical will be toxic to animals and humans – The Ames test to assess DNA damage caused by carcinogens – Gene microarrays to determine target genes

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 • LD 50 is a measure of acute toxicity – LD 50 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 LD 50, 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.

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 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.

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:

Biohazard Safety

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 Classification • The National Institutes of Health (NIH) has determined a classification system for biohazardous agents based on Risk Groups (RG). Risk Group 1 (RG 1) Agents that are not associated with disease in healthy adult humans Risk Group 2 (RG 2) Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available Risk Group 3 (RG 3) Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk) Risk Group 4 (RG 4) Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk)

Biological Hazards • Sterilization techniques are generally effective in destroying biohazard agents (except prions and spores) – Autoclave – Bleach – Alcohol • Biohazards are another reason (in addition to avoiding ingestion of toxic chemicals) for good lab hygiene – Glove use and proper gloves hygiene – Hand washing – No food or drink in lab

Other Biochemistry Safety Concerns

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. Keep the -80° – Handle glass dewars with caution – danger of freezer closed! exploding glass if they are knocked over and broken.

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.

GHS Symbols

Liquid Nitrogen Safety • Liquid nitrogen (LN 2) is commonly used to rapidly freeze proteins and bacteria • LN 2 rapidly evaporates and can displace air in enclosed spaces causing suffocation • LN 2 can cause death of human tissue from extreme cold • Minor contact can cause “burns” • Evacuated glass dewars can sometimes burst unexpectedly • LN 2 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 LN 2 in a closed automobile DON’T transport LN 2 in a passenger elevator DON’T allow a storage dewar to tip over DON’T leave cold fingers on a vacuum line in LN 2 overnight • DON’T use without PPE! • • • NO!

Liquid Nitrogen Do’s YES! • DO use or dispense LN 2 only in well ventilated areas • DO ensure glass dewars are taped or wrapped • DO use approved containers only such as a dewar or NO! threaded cryovials for storage • DO make sure any vessel with LN 2 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 YES! • Closed toed Shoes • Long pants

Special Note on LN 2 transport for Chemistry Magic Shows Use only a sturdy LN 2 dewar Keep windows of car open Do not put LN 2 next to any passenger in the car Secure the LN 2 such that it cannot tip over (use seatbelt, other heavy, bulky objects to block it in) • Be prepared to leave the vehicle if a spill occurs • Do NOT allow K-12 students to touch LN 2 • Do NOT allow K-12 students to approach your glass dewar without goggles • •