1. Biochemistry Lab Safety

2. Personal Protective Equipment: What must be worn when you work in the laboratory. 2 1. PPE Eye Protection Lab Coat Long Pants Closed Toed Shoes – no exposed skin around feet Lab gloves – when required

3. 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

4. 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.   Clothing and Foot Protection 4 

5. Use of Gloves Remove gloves before handling objects such as doorknobs, telephones, pens, computer keyboards, pH meter or other electronic buttons, or phones 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 anytime you take them off. You should expect to use several pairs of gloves in any given lab period. Glove video 5

6. 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

7. 7 Eye Wash

8. Safety Shower 8

9. Using the Fume Hoods properly 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. This window/bar is called the sash. 9

10. × ✓ ✓✓ Closed, not in use In use, side-to-side panel used as shield In use, sash (window) raised to less than 18 inches Don’t open side shields to make one big window. 10

11. 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

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

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

14. 14

15. Types of Fire Extinguishers 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. 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. 15

16. 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

17. 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 things in the floor because someone will trip over it. Don’t leave cords dangling because someone will trip over them. 17

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

19. Centrifuge Safety

20. 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 Swinging Bucket rotor Disposable tubes Need adaptors Fixed angle rotor

21. 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

22. 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) Both knobs are tightened in some models to secure rotor to the spindle Counterbalance your labeled sample

23. 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

24. Safety Overview http://www.youtube.com/watch?v=q_0phA03 4n0 http://www.youtube.com/watch?v=q_0phA03 4n0 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.

25. Autoclave Safety 25

26. 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.

27. 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

28. 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

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

30. 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

31. 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

32. 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

33. Autoclaving Waste 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 This bag is too full

34. 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

35. 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

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

37. Overview http://www.youtube.com/watch?v=T901F2W 7wks http://www.youtube.com/watch?v=T901F2W 7wks 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.

38. Toxic Chemical Safety

39. 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 Corrosives Irritants, sensitizers, acutely toxic

40. 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

41. 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.

42. 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.

43. Toxic ChemicalsIn 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.

44. 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

45. Acrylamide Acrylamide is the monomeric precursor to polyacrylamide used in SDS-PAGE. Avoid working with the powder due to inhalation hazard (instead work with solutions) Use gloves when handling Polymerize excess solution for safer disposal 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.

46. Sodium Azide Sodium azide is commonly found in dilute solutions to prevent bacterial growth. 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. The acute toxicity of sodium azide is high

47. Phenylmethanesulfonyl Fluoride PMSF is used in solution to inhibit proteases 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 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.

48. 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.

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

50. Look for the health NFPA category rated from 0-4, and read warning statements. Toxic Chemicals and Material Safety Data Sheets (MSDS) 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

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

52. Biohazard Safety

53. 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.

54. Biohazard Classification The National Institutes of Health (NIH) has determined a classification system for biohazardous agents based on Risk Groups (RG). Risk Group 1 (RG1) Agents that are not associated with disease in healthy adult humans Risk Group 2 (RG2) Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available Risk Group 3 (RG3) 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 (RG4) 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)

55. 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

56. Other Biochemistry Safety Concerns

57. 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.

58. 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. Neal Tonks – 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.

59. 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.

60. GHS Symbols

61. 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

62. 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!

63. Liquid Nitrogen Do’s 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 YES! NO! YES!

64. Special Note on LN2 transport for Chemistry Magic Shows Use only a sturdy LN2 dewar Keep windows of car open Do not put LN2 next to any passenger in the car Secure the LN2 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 LN2 Do NOT allow K-12 students to approach your glass dewar without goggles

65. 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. Neal Tonks– 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.