1. Revised August 2019
CHEM 355 Lab Safety

2. 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

3. 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.   

4. 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

5. 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.

6. 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.

7. Eye Wash

8. Safety Shower

9. 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.

10. 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

11. 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.

12. ✓ ✓ × ✓ 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. × ✓

13. 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.

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

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

17. 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.

18. 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

19. 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 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.

20. Centrifuge Safety

21. 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

22. 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

23. 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

24. 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

25. 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.

26. Autoclave Safety

27. 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.

28. 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

29. 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

30. 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.

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

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

38. 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 Dr. Stephanie Boussert

39. Other Biochemistry Safety Concerns

40. 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

41. GHS Symbols

42. 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.

43. 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

44. 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.

45. 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

46. 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

47. 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.

48. 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.

49. 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*

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

51. 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

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

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.
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)

54. 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.

55. 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!

56. 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

57. 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!

58. 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!

59. 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.