Safety Training Information and
Standard Operating Protocols
LABORATORY NAME
Arizona State University
DEPARTMENT NAME
Effective Date
Note: Review must occur at least annually, when updates are added, or as needed.
By signing below you agree to the following statement:
I have read the procedures contained in this plan. I understand all procedures contained
in this plan and agree to comply with them at all times. I have been given an opportunity
to ask questions and all of my questions have been answered.
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LAB NAME CONTACT INFORMATION
Research
Name
Position
Email
Phone
Facility Managers, Support Staff and Safety Contacts
Name
Position
Email
Phone
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1. Introduction and general policies
The [Insert] Lab Name research group at ASU is a [Insert] Lab Type. [Insert] Lab
Description. We are classed as a Biosafety Level [Insert] Number (BSL [Insert]
Number) laboratory. This biosafety containment level is determined by CDC/NIH
Guidelines. [Insert] Biosafety Level Specifications. Our National Fire Prevention
Association (NFPA) class is “[Insert] NFPA Ratings, each separated by ‘-‘. E.g. 4-43-W ” and is explained below, as well as in Section 1.1.5.
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4
3
W
Blue = health and toxicity information
4 = Very short exposure could cause death or major residual injury
Red = flammability
4 = Will rapidly or completely vaporize at normal atmospheric
pressure and temperature, or is readily dispersed in air and will burn
readily. Flash point below 23C (73F).
Yellow = reactivity
3 = Capable of detonation or explosive decomposition but requires a
strong initiating source, must be heated under confinement before
initiation, reacts explosively with water, or will detonate if severely
shocked.
White = other possible hazards
W is used to denote compounds that are highly reactive with water.
The following guidelines and SOPs should help explain normal laboratory operations, as
well as provide guidance in specific situations. If you have questions or concerns about
ANY safety issue, please contact [Insert] Responsible Person’s Name(s) (Position).
1.1. General notes about maintaining a safe workspace
1.1.1. Securing the space
The laboratory should be kept locked whenever there is no one present. This includes
daytime hours if you intend to be elsewhere in the department or on campus.
1.1.2. Laboratory clutter
It is everyone’s responsibility to maintain a safe and clutter-free lab environment. Fire
code requires maintaining a clear passageway of at least 4 feet. Piles of cardboard or
garbage larger than what will fit in the standard trash must be removed promptly.
Personal belongings should be kept in appropriate locations and not stored on the
counters or floors.
1.1.3. Food and drink in the laboratory space
Absolutely no food or drink is permitted in the lab.
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1.1.4. Electrical equipment
Periodically check all electrical equipment to ensure that all cords and connections are
intact. Do not use anything with frayed or damaged electrical components! If a
component is damaged, you make take it to [Insert] Responsible person’s name(s)
and [Insert] if exists, location of the repair shop.
1.1.5. Required Safety Training
All employees who are paid by ASU whether it is through regular payroll or through
grants or stipends must have the following training:
Fire Safety and Prevention-Initial and annual refresher
Laboratory Safety-Initial and annual refresher
Hazardous Waste Management-One time training
nitial training must be attended in person however refreshers are available on line via
BlackBoard. Hazardous Waste Management is also available on Blackboard. Go to the
EH&S website to register for classes http://uabf.asu.edu/ehs_training.
1.2. Labeling & storage of chemicals, solutions and samples
All chemicals, solutions and samples must be clearly labeled with the content, full name
and the NFPA (National Fire Prevention Agency) hazard diamond. NFPA hazard
diamond is the “Standard System for the Identification of the Hazards of Materials for
Emergency Response”. This classification system uses a series of numbers from 0-4 to
denote chemical hazards in four classes. If the chemical is a stock chemical (i.e.
purchased from a vendor) it should have the appropriate information already on the
label. If not, or if the chemical is a homemade stock or buffer, a label with the required
information must be affixed. The following image and table should help explain the
meaning and use of the NFPA classifications.
Blue = health and toxicity information
Red = flammability
Yellow = reactivity
White = other possible hazards
Figure 1.2 – NFPA hazard diamond.
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An explanation of each of the numbers in each hazard class is detailed below.
Blue (health)
0
1
2
3
4
Material that on exposure would offer no hazard beyond ordinary material.
Material that on exposure would cause irritation but only minor residual injury.
Material that on intense or continued but not chronic exposure could cause
temporary incapacitation or possible residual injury.
Material that on short exposure could cause serious temporary or residual injury.
Material that on very short exposure could cause death or major residual injury.
Red (flammability)
0
1
2
3
4
Material will not burn.
Material must be preheated before ignition can occur.
Material must be moderately heated or exposed to high ambient temperature for
ignition to occur.
Liquids and solids that can be ignited under ambient temperature conditions.
Materials that will rapidly or completely vaporize at atmospheric pressure and
normal ambient temperature, or that are readily dispersed in air and that will burn
readily.
Yellow (reactivity)
0
1
2
3
4
Material that in itself is normally stable, even under fire exposure conditions and is
not reactive with water.
Material that in itself is normally stable but which can become unstable at
elevated temperatures and pressures.
Material that readily undergoes violent chemical change at elevated temperatures
and pressures or which reacts violently with water or which may form explosive
mixtures with water.
Material that in itself is capable of detonation or explosive decomposition or
reaction but requires a strong initiating source or which much be heated under
confinement before initiation or which reacts explosively with water.
Material that in itself is readily capable of detonation or explosive decomposition
or reaction at normal temperatures and pressures.
White (other hazard information)
At the bottom of the NFPA diamond is a white space used for additional information
about the chemical or material. Examples are: W indicating reactivity with water, COR
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for corrosives, RAD (radiation), OXY (oxidizer), and CARC for carcinogen. When in
doubt, ask or look it up within a reference!
1.2.1. All carcinogens and toxic chemicals must be labeled as such!
Any chemicals with special hazards must be labeled as such. This includes any buffers
or solutions made with said chemicals in ANY proportion.
1.2.2. Secondary containment policies
All reactions involving corrosive or potentially hazardous chemicals must be performed
with the safeguard of a secondary container. If any part of a laboratory experiment must
be transported to another area that crosses any point of public access such as a
hallway or elevator, clean, closed secondary containment must be used to transport the
material. Remove PPE including gloves before leaving the laboratory and when in
public areas. Even if the PPE is clean, the perception is that it is not and you are
therefore contaminating public areas. [Insert] Details, if any.
1.3. Non-chemical Hazards
1.3.1. Sharps
All sharps (syringes, razor blades, etc.) must be disposed of after use in a red sharps
container. Razor blades may be reused only if they are stored with the blade fully
protected in a piece of Styrofoam. Needles must not be recapped unless you have
received documented training using the one-handed scoop method (see SOP on page
19). Syringes must be then disposed of along with needles and not disconnected.
Note: Plastic pipette tips such as those used in automatic pipettors (such as Eppendorf
type pipettes) must be placed in sharps containers. They cannot go in the regular trash.
Alternately they can be placed in red biohazard bags and autoclaved. Custodial staffs
have no way of telling if these pipette tips were used for biohazardous materials,
chemically hazardous materials or water. These tips can poke through trash can liners
and cause an injury that breaks the skin.
1.3.2. Broken glassware
Empty glassware of hazardous materials, then dispose of the glass in the ASU “broken
glassware” yellow color coded metal can or a box lined with an impermeable liner.
When the “broken glassware” collection container is full, it can be collected by ASU
Custodial Services. Any questions regarding hazardous materials should be directed to
EH&S.
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1.4. Working in the hoods
1.4.1. Fume hoods
Fume hoods are designed for temporary chemical storage (preparation of chemicals,
temporary reaction setups, etc.) and not for long-term storage. Waste bottles may be
temporarily stored in the fume hood until full and ready to be picked up by EH&S (see
Section 1.5 on the Generation and Storage of Waste).
1.4.2. Laminar flow hoods
Laminar flow hoods are designed to maintain a sterile working environment inside the
hood. They are not designed to protect the operator from any chemical hazards.
1.5. Personal Protective Equipment (PPE)
1.5.1. Contact lenses in the laboratory
Contacts may not be worn in the laboratory. Gas permeable lenses are a safety risk. If
corrective lenses are required, safety glasses must be worn in addition to corrective
lenses.
1.5.2. Safety glasses
Safety glasses must be worn at all times, by all staff and while performing any tasks
while in the laboratory. Regular or prescription glasses are not considered safety
glasses unless they also have side shields. NO EXCEPTIONS!
1.5.3. Gloves
Gloves should be used when appropriate for a task. Latex gloves do not provide
protection from the hazards involved with some types of chemicals. Familiarize yourself
with the MSDS sheets for the chemicals you are working with. Gloves must be removed
before touching any computers or shared instrumentation. Do not touch door handles
with dirty gloves! If you must carry an item between laboratories, use a clean, closed
secondary container for transport. Remove PPE including gloves when leaving the
laboratory and entering public areas such as hallways and elevators. Remember, even
if your PPE is clean, public perception is that they are dirty and that you are
contaminating public areas.
1.5.4. Appropriate attire
Appropriate safety attire must be worn at all times. Closed-toed shoes are required at
all times while in the laboratory environment. This includes instances of “just doing
computer work” and “just stopping by to pick something up.” If you have open-toed
shoes on, you cannot enter the laboratory for any reason. Lab coats with long sleeves
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are required when working with chemicals or any type or strength. Shorts are not
appropriate when working with any potentially hazardous material, especially chemicals.
1.6. Material safety data sheets (MSDS) & chemical hazards
1.6.1. Location of MSDS
[Insert] MSDS sheets location details.
Acids
(oxidizing)
Bases
(alkalis)
X
X
Oxidizers
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Poisons
(inorganic
)
Oxidizers
Bases
(alkalis)
Acids
(oxidizing)
X
Water
Reactives
X
X
Solvents
(organic)
Acids
(organic)
X
Poisons
(organic)
Acids
(inorganic)
Acids
(organic)
Acids
(inorganic
)
1.6.2. Chemical storage
Chemicals must be stored with respect to compatibility issues. General chemicals may
be stored on the shelf, provided they are properly labeled with the full name of the
chemical and the appropriate NFPA hazard information. For storage of chemicals
reference the chart below (X = incompatible).
X
X
Poisons
(inorganic)
X
X
X
Poisons
(organic)
X
X
X
X
X
X
Solvents
(organic)
X
X
X
X
X
Water
Reactive
X
X
X
X
X
X
X
Acids
 Segregate acids from reactive metals such as sodium, potassium, and magnesium.
 Segregate oxidizing acids from organic acid and flammable and combustible
materials.
 Store acetic acid as a flammable liquid. This is an organic (carboxylic) acid that will
react if it comes in contact with an oxidizing acid.
 Nitric acid and hydrochloric acid may be stored in the same corrosive storage
cabinet, but they must be kept in separate drip trays. DO NOT MIX WASTE
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

CONTAINING THESE TWO CHEMICALS! They can combine to form chlorine and
nitrosyl chloride gases—both are toxic.
Segregate acids from chemicals that could generate toxic or flammable gases upon
contact, such as sodium cyanide, iron sulfide and calcium carbide.
Segregate acids from bases.
Bases
 Segregate bases from acids, metals, explosives, organic peroxides and easily
ignitable materials.
 Do not store aqueous sodium and potassium hydroxide solutions in aluminum drip
trays. These bases will corrode aluminum.
Solvents (flammable and combustible liquids)
 Store in approved safety cans or cabinets.
 Segregate from oxidizing acids and oxidizers.
 Keep away from any source of ignition: heat, sparks, or open flames.
Oxidizers
 Keep away from combustible and flammable materials.
 Keep away from reducing agents such as zinc, alkali metals, and formic acid.
Cyanides
 Segregate from aqueous solutions, acids and oxidizers.
Water-Reactive Chemicals
 Store in a cool, dry place, away from any water source.
 Make certain that a Class D fire extinguisher is available in case of fire.
Pyrophoric Substances
 Pyrophoric substances can spontaneously ignite in air
 If in original container store in a cool, dry place, making provisions for an airtight
seal.
 Store in a glove box after the material has been opened.
Light-Sensitive Chemicals
 Store in amber bottles in a cool, dry, dark place.
Peroxide-Forming Chemicals
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

Most peroxide forming chemicals are also flammable liquids. Therefore, store in
airtight containers in a flammable storage locker. Date the bottle when it is received
and dispose of when 6 month shelf life is exceeded. Check bottles for expiration
dates.
Segregate from oxidizers and acids.
Toxic Chemicals
 Store according to the nature of these chemicals, using appropriate security where
necessary.
ETHIDIUM BROMIDE
Ethidium bromide is a large, flat basic molecule that resembles a DNA base pair.
Because of its chemical structure, it can intercalate (or insert) into a DNA strand.
Ethidium bromide is commonly used in molecular biology laboratories to stain
electrophoresis gels. The compound forms fluorescent complexes with nucleic acids
and these can be viewed under UV light.
Ethidium bromide is a known mutagen in certain animal and microorganism test
systems. Although the compound has not been thoroughly evaluated in humans,
based on current toxicity data and its interaction with DNA it should be handled
with considerable caution.
Chemical Name: Ethidium Bromide Synonyms: Ethobromide; Dromilac; Homidium
Bromide
Chemical Formula: C21H20N3Br MW: 394.4; CAS Number: 1239-45-8 EtBr; RD
1572
Description: Compound may be ordered as a solid powder, tablets, or a stock solution
of known concentration. Melting Point (solid): 500o F (260o C)
Exposure Limits
Occupational exposure limits have not been established by OSHA, NIOSH, or ACGIH.
Acute Animal Toxicity
Oral; rat: LD50 = 1503 mg/kg
ppm
Subcutaneous; mouse: LD50 = 110 mg/kg
20 mg/kg
Inhalation; rat LC50 = 0.0118-0.1340
Intraperitoneal; mouse: LDLo =
Personal Protective Equipment (PPE)
Gloves: Wear Nitrile gloves to prevent hand contamination. Thin disposable gloves
(such as 4, 6, or 8 mil blue Nitrile gloves) used in laboratory operations provide a
contact barrier only and should be disposed of immediately when contamination is
suspected. Disposable gloves should not be worn for protection from hazardous
chemicals without double gloving because of the potential for pinholes. Latex
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disposable gloves are especially prone to defects and pinholes and are not
recommended.
Glasses: Wear chemical safety glasses with side shields.
Lab Attire: Always wear long pants, closed toe shoes, and a lab coat when handling
hazardous materials.
Health Hazard Data
Acute – This material may be harmful by all routes of entry; inhalation, ingestion, or skin
absorption. It may cause eye and skin irritation and is irritating to mucous membranes
and upper respiratory tract.
Chronic systems (yeast cells). The chemical, physical, and toxicological properties have not
been thoroughly investigated in humans.
Emergency and First Aid Procedures
In the event of any chemical exposure emergency, dial 911 to reach the Campus
dispatch and follow the instructions on the Emergency Response Guide.
Decontamination & Disposal Procedures
Dispose of all ethidium bromide waste through the campus Hazardous Waste
Department. Drain disposal of ethidium bromide solutions is NOT ALLOWED. Disposal
of dried gels containing ethidium bromide or any contaminated solid waste (gloves,
microfuge tubes, etc.) in the general campus waste stream is NOT ALLOWED.
Collection and detoxification of ethidium bromide solutions for drain disposal is NOT
ALLOWED.
Decontamination of Spills
Detoxification and decontamination of ethidium bromide by oxidation with household
bleach previously recommended has shown that this produces compounds that are
more hazardous than the original ethidium bromide compound. Therefore, OXIDATION
WITH BLEACH IS NOT AN ACCEPTABLE DESTRUCTION TECHNIQUE and is not to
be used.
Method 1 (Lunn and Sansone 1989) Recommended technique for smaller, isolated
spills of dilute solutions.
1. Turn off all electrical equipment before decontamination. Wear appropriate protective
equipment including gloves, lab coat, and safety goggles.
2. Absorb all free liquid from the spill with paper towels. Caution: The towels may
contain levels of concentrated ethidium bromide and should be handled appropriately.
3. Scrub the contaminated surface six separate times, each time using a fresh, wet
paper towel. Place all towels into a container. Collect all paper towels and debris
involved with the clean up and dispose of as hazardous waste through EH&S.
4. Dry the equipment or surface and place into service.
Contact EH&S for larger, more diffuse spills.
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1.7. Accidents, Fire and Evacuation
1.7.1. In the event of an accident
Take appropriate first aid action, and then secure the area. Go to a medical center to
receive appropriate treatment. Report to the PI ([Insert] Responsible Person’s
Name), who will then take care of the necessary paperwork. If a spill kit is used, it must
be immediately reported to the PI ([Insert] Responsible Person’s Name), the
Departmental Safety Officer ([Insert] Responsible Person’s Name if applicable) and
call EH&S Hazardous Materials Response at extension 5-3534.
If the accident is related to the building (leaks, etc.) you may contact [Insert]
Responsible Person’s Name (Position) on his cell phone at [Insert] Contact
number.
1.7.2. In the event of an emergency
Call 911! Then secure the area. Immediately contact the PI ([Insert] Responsible
Person’s Name), followed directly by the Safety Officer ([Insert] Responsible
Person’s Name). If PI deems it necessary, [Insert] Responsible Person’s Name
(Facility Manager) may also be contacted to deal with any infrastructure issues.
1.7.3. Fire extinguishers
A fire extinguisher is located at the east entrance door to the lab. In the event of a fire,
use the PASS technique (Pull, Aim, Squeeze, and Sweep). Everyone in the lab should
have taken (and be up-to-date on) their fire safety training. Annual refreshers are
required. You may take the ½ hour class on Blackboard.
1.7.4. Eyewashes and safety showers
Eyewashes must be flushed once a month. Be sure to document that this has been
done. Safety showers are checked annually by EH&S. Employees who may be
exposed to hazardous materials shall be instructed in the location and proper use of
emergency showers and eyewashes (American National Standard for Emergency
Eyewash and Shower Equipment ANSI Z358.1-2004.)
1.7.5. Spill kit
A chemical spill kit is available [Insert] location of spill kit. If the spill kit is used,
[Insert] Responsible Person’s Name(s) and EH&S Hazardous Waste Management
must be contacted at once. Call 5-3534 to notify EH&S.
1.7.6. Evacuation and escape plans
In the event that an evacuation is required, please follow the plans below:
[Attach] Building plan diagram
[Insert] Primary Area of Rescue Assistance and details.
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[Insert] Secondary Area of Rescue Assistance and details.
[Insert]Procedure to evacuate facility.
1.8. Generation and temporary storage of waste
ASU has two main policies on the generation and storage of hazardous waste:
(1) No waste can be disposed of on-site (nothing in the trash, nothing down the drain.)
(2) The waste generator assumes cradle-to-grave responsibility for the waste.
Taken together, these mean that ALL waste and byproducts generated in the [Insert]
Laboratory Name (By product and waste details) must be classified and segregated
for waste processing by ASU EH&S Hazardous Waste Management. In regards to
hazardous substances and waste, this means NOTHING goes down the sink, and
NOTHING goes in the garbage unless it is pure water or uncontaminated waste
respectively. All generated waste is processed through EH&S Hazardous Waste
Management. [Insert] Location of lab hazardous waste collection area. When
waste is generated it must be disposed of in the appropriate bottle and the contents
noted on the corresponding waste composition data sheet. Note that all waste
containers must have secondary containment (a tray underneath). When a waste bottle
is full, EH&S may be contacted at 480.965.3899 or a hazardous waste pickup request
made online at http://www.asu.edu/uagc/EHS/waste.htm. At this time, waste disposal
data should be logged in the “Waste Disposal Log”, located adjacent to the MSDS
sheets on [Insert] MSDS Sheets Location. Even if a waste bottle is not full, it should
not be left in the laboratory for more than one year.
Please consult the following list for data on disposal of different types of waste:
Aqueous waste
Aqueous waste may not go down the sink! Not even simple salt solutions! Dispose of
aqueous waste in the appropriate bottle, ensuring that there are no reactivity or
incompatibility issues with the already disposed-of items. When in doubt, look it up or
start a new waste bottle! Be aware that bleach-containing solutions are often highly
reactive and can form toxic reaction products with other chemicals. Dispose of bleach
separately.
Biohazard bins
Small red biohazard bins are for accumulation of bacterial and algal plates (with or
without growth) or bacterial or algal contaminated tips ONLY. These items will be
autoclaved in-house then transferred to the large red biohazard bin for pickup by EH&S.
Any other items should be directly placed in the large biohazard bin. When the large
red biohazard bin is approx. 75% full or weighs about 60lbs, contact EH&S for a pickup
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at 480.965.3899 or make a hazardous waste pickup request online at
http://www.asu.edu/uagc/EHS/waste.htm.
Ethidium bromide (EtBr) gels and EtBr-containing buffer
EtBr-containing gels may be disposed of in the dedicated 5-gallon pail provided by
EH&S. Ethidium bromide- containing buffers should be discarded in the dedicated EtBr
waste bottle in the [Insert] Location. When full, call for pickup as with any other waste.
General trash
Items acceptable in general trash include paper (but should try to recycle) and plastic
(packaging, etc.). Nothing that has touched chemicals should go in the general trash.
Custodial generally comes through about 3x a week to pick up trash. If there are issues
with trash pickup, you may contact custodial services 480.965.9633 (Facilities
Management Service Centre).
Liquid cultures of algae and bacteria
Bacterial waste under ~10 mL may be treated with an equal volume of 10% sodium
hypochlorite solution. Allow it to sit for 1 hour in the hood and then dispose of in the
dedicated “Bleach-containing waste” container.
Algal cultures of small volumes (under 1 L) should be poured into the Waste container.
The initial culture vessel should be rinsed 2x with water, with the waste water also
transferred into the Waste container. The initial culture flask should be rinsed once with
10% sodium hypochlorite solution, with those rinsings going into the “Bleach-containing
waste”. Two more rises with water (waste going into the “Bleach-containing waste”
container) sufficiently decontaminates the flask. It may now be washed with soap and
water and put back into normal use.
When the Waste container is ¾ full, it should be autoclaved to deactivate the contents.
An autoclave cycle of 45 minutes sterilization and 45 minutes exhaust should be
sufficient. After the decontaminated solution has cooled to room temperature, it should
be poured into the Culture Biowaste Drum provided by EH&S.
Large volumes of algal or bacterial culture should be deactivated prior to disposal by
autoclaving in the original culture flask. Once cooled, the deactivated solution can also
be poured into the Culture Biowaste Drum provided by EH&S. Two rinses with water,
with rinsings going into the drum as well, are sufficient to decontaminate the flask. It
may then be washed with soap and water and will be ready for further use.
For protocols that generate culture supernatant as a result of centrifuging cell cultures,
the supernatants must be treated as a recombinant species. Small volumes (as in
plasmid minipreps) may be treated with bleach as in the first section. Larger volumes of
supernatant (as when doing a TK prep) should be poured into the 6L culture flasks
originally used to grow the strains then autoclaved. Once cool, the solution should be
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poured into the EH&S provided Culture Biowaste Drum, rinsed two times as above, then
cleaned and used as usual.
Organic solvents
Organic solvents are segregated into halogenated and non-halogenated bottles for ease
of recycling. As most solvents are highly flammable, keep this in mind and keep the
waste bottle away from any sources of ignition!
Sharps
Used sharps (syringes, needles, razor blades, etc.) must be disposed of in the small
sharps container located [insert] location. After use, the needle must not be recapped.
Exceptions to this policy are allowed only if re-sheathed occurs using the one-handed
scoop method. Safety training using this method must be documented (see SOP page
19.) EH&S Hazardous Waste Management will pick up the bin when full – call 5-3534 or
arrange online at http://uabf.asu.edu/waste_form.
Pipette Tips and other waste
Plastic pipette tips used for any reason must be disposed of in the EH&S red biowaste
bin for autoclaving or a sharps container. A convenient way to do this is to have a small
(1 L) plastic beaker on the workbench for disposal while you work. At the end of the day
or when the beaker is full, dispose of it in the EH&S biowaste bin (red). Gloves may go
in the trash provided they have not contacted hazardous chemicals or biologicals.
2. Specific Standard Operating Procedures
(SOPs)
Some processes require additional training and/or notes to ensure safety. The following
should help you complete the listed procedures safely and efficiently.
2.1. Autoclaving
Autoclaves are a commonly used piece of equipment in molecular biology labs.
However, they do present a serious hazard in terms of heat, steam and pressure, as
well as biological hazards. As many autoclaves differ, ensure that you are familiar with
safe operating guidelines. When in doubt, ASK! Some good guidelines:


A standard cycle for liquids is 45 minutes for sterilization, 35 minutes for exhaust. A
dry run requires far less exhaust.
Do not autoclave items containing corrosives (e.g. acids, bases, phenol), solvents or
volatiles (e.g. ethanol, methanol, chloroform) or radioactive materials.
15









Before using the autoclave, check inside the autoclave for any items left by the
previous user that could pose a hazard (e.g. sharps).
Load the autoclave properly as per the manufacturer’s recommendations.
To prevent bottles from shattering during pressurization, the caps of containers with
liquids must be loosened before loading.
Use a tray with a solid bottom and walls to contain the contents and catch spills – if it
is plastic, ensure that it is autoclave-safe!
Add 1/4 to 1/2 inch of water to the tray so the bottles will heat evenly.
Individual glassware pieces should be within a heat resistant plastic tray on a shelf
or rack and never placed directly on the autoclave bottom or floor.
Make sure the door of the autoclave is fully closed, that the correct sterilization and
exhaust times have been chosen, that the correct cycle (fluid or regular) is chosen,
and (if necessary) that the steam generator is on or open.
Before opening the autoclave door after a cycle, ensure that the chamber pressure
is at zero. Stand to the side when opening the chamber to prevent a steam burn.
When removing items from an autoclave, goggles and heat-resistant gloves should
be worn.
Bleach
Sodium hypochlorite (bleach) is used in a 10% solution with water to decontaminate
benches and to deactivate recombinant organisms prior to their disposal. This solution
weakens and should be made fresh every 24 hours. Sodium hypochlorite is harmful if
swallowed or inhaled, causes irritation to eyes and respiratory tract as well as
substantial but temporary eye injury on contact. Use the appropriate PPE (goggles,
gloves and preferably a lab coat or apron) when working with bleach. To deactivate
recombinant organisms, see 2.11 – Working with recombinant organisms.
When disposing of bleach-containing solutions, be sure to use a container labeled
“Sodium hypochlorite / Bleach waste ONLY.” Be VERY careful to make note of any
possible incompatibilities and reactivities: ammonia (toxic chloramine gas may evolve),
amines, ammonium salts, aziridine, methanol, phenyl acetonitrile, cellulose,
ethyleneimine, oxidizable metals, acids, soaps, and bisulfates.
Be aware that the Qiagen Plasmid Miniprep kits contain chemicals that are
incompatable with bleach! This is a good reminder to check the compositions of ALL kit
reagents prior to use. CHECK REACTIVITIES BEFORE YOU DISPOSE OF
CHEMICALS!
Centrifuges
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Centrifuges are a common and powerful laboratory tool, but they can also be very
dangerous. Ensuring that the following safety guidelines are met should prevent most
issues from arising.
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Centrifuge tubes must be balanced at all times! With slower table-top centrifuges,
this may be done by volume, but with high speed and ultracentrifuges, tubes must
be balanced to no greater than 100 and 10 mg differences respectively.
Make sure the lid is on the rotor and that it is properly secured.
Ensure that the rotor is properly secured to the drive.
Do not exceed the rotor’s maximum mass or maximum speed.
Do not run swinging bucket rotors with missing buckets.
Make sure that hooked buckets are securely fastened and can swing freely.
Use only centrifuge tubes that are rated for the speed you want to use.
Examine tubes and bottles for cracks or stress marks before using them.
Do not overfill centrifuge tubes as leakage may occur when tubes are filled to
capacity. The maximum capacity for centrifuge tubes is 3/4 full.
Always cap tubes, preferably with screw-top lids.
If something goes wrong with the centrifuge:
 Stop the centrifuge immediately if you hear unusual noises or vibrations.
 Turn off the centrifuge and do not attempt to open the lid right away. If there is a
leak, you will want to allow any generated aerosols to settle.
 If you have been centrifuging anything considered a health hazard, keep the lid
closed until you have planned out a decontamination protocol.
 If there is any leakage, clean it up as required by the safety class of the components.
If the spill is not a safety hazard, the rotor should be cleaned with a mild, nonabrasive detergent rinsed with DI water and dried well. The cavity should be cleaned
and dried as well.
 If damage is apparent, contact the Lab Coordinator or PI to determine who to call for
centrifuge repairs.
2.2. Cryogens
[Insert] details of any cryogens in the lab. Use exercise extreme caution to prevent
cold burns!
Some points to remember for liquid nitrogen:
 Face shields and goggles should be worn during the transfer and normal handling of
liquid nitrogen or other cryogenic materials.
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Loose fitting, heavy leather, or other insulating protective gloves should be worn at
all times when handling cryogenic fluids. Protection for exposed skin should be
worn to prevent injuries from liquid spraying or spilling.
Some articles of clothing such as sweaters or scarves can absorb liquid nitrogen
readily and hold it next to the skin. Always use appropriate PPE!
All uses of cryogens must be in a well-ventilated area to prevent possible gas or
vapor accumulation (note that the volumetric expansion ratio of liquid to atmospheric
nitrogen is nearly 700 to 1 – this can result in the rapid generation of an oxygen
deficient atmosphere and possible asphyxiation).
Transfer operations involving open cryogenic containers such as Dewars, must be
conducted slowly to minimize boiling and splashing of the cryogenic fluid. Perform
all transfers below the chest level of the person pouring the liquid.
Liquid nitrogen should only be stored or used in containers and systems specifically
designed for cryogenic liquids and handled in accordance with applicable standards,
procedures, or proven safe practices.
In transporting liquid nitrogen, be very cautious not to endanger yourself or others in
the area.
Because it is an asphyxiant, liquid nitrogen or other cryogenic materials may be
transported in an elevator only when using the following protocol:
o An operator will load the cryogenic container into the elevator and start the
car. The operator will not accompany the car to its destination.
o An operator will be stationed on the floor to where the liquid nitrogen is being
transported to receive the cryogenic container.
o After the cryogenic container is removed from the elevator, the elevator can
be returned to normal service.
Dry Ice
 Wear heavy gloves while using to avoid cold burns.
 Transport only in approved containers.
 Dry ice also presents an asphyxiation risk (subliming to CO2) – use only in well
ventilated rooms.
2.3. Ethidium Bromide
Ethidium bromide is a large, flat basic molecule that resembles a DNA base pair.
Because of its chemical structure, it can intercalate (or insert) into a DNA strand.
Ethidium bromide is commonly used in molecular biology laboratories to stain
electrophoresis gels. The compound forms fluorescent complexes with nucleic acids
and these can be viewed under UV light.
Ethidium bromide is a known mutagen in certain animal and microorganism test
systems. Although the compound has not been thoroughly evaluated in humans,
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based on current toxicity data and its interaction with DNA it should be handled
with considerable caution.
 Minimize the area in which ethidium bromide is to be used
 Designate an area in the lab that is to be used only when working
with ethidium bromide.
 Outline or otherwise mark this area by some useful method such as
red tape to limit unnecessary exposure to ethidium bromide
 Post a sign in the area with appropriate warnings
Chemical Name: Ethidium Bromide Synonyms: Ethobromide; Dromilac; Homidium
Bromide
Chemical Formula: C21H20N3Br MW: 394.4; CAS Number: 1239-45-8 EtBr; RD
1572
Description: Compound may be ordered as a solid powder, tablets, or a stock solution
of known concentration. Melting Point (solid): 500o F (260o C)
Exposure Limits
Occupational exposure limits have not been established by OSHA, NIOSH, or ACGIH.
Acute Animal Toxicity
Oral; rat: LD50 = 1503 mg/kg
Inhalation; rat LC50 = 0.0118-0.1340
Ppm
Subcutaneous; mouse: LD50 = 110 mg/kg
Intraperitoneal; mouse: LDLo =
20 mg/kg
Personal Protective Equipment (PPE)
Gloves: Wear Nitrile gloves to prevent hand contamination. Thin disposable gloves
(such as 4, 6, or 8 mil blue Nitrile gloves) used in laboratory operations provide a
contact barrier only and should be disposed of immediately when contamination is
suspected. Disposable gloves should not be worn for protection from hazardous
chemicals without double gloving because of the potential for pinholes. Latex
disposable gloves are especially prone to defects and pinholes and are not
recommended.
Glasses: Wear chemical safety glasses with side shields. Regular or prescription
glasses are not considered eye protection unless there are side shields on the frames.
Lab Attire: Always wear long pants, closed-toed shoes, and a lab coat when handling
hazardous materials such as chemicals.
Health Hazard Data
Acute – This material may be harmful by all routes of entry; inhalation, ingestion, or skin
absorption. It may cause eye and skin irritation and is irritating to mucous membranes
and upper respiratory tract.
Chronic ds and was mutagenic in a number of test
systems (yeast cells). The chemical, physical, and toxicological properties have not
been thoroughly investigated in humans.
Emergency and First Aid Procedures
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In the event of any chemical exposure emergency, dial 911 to reach the Campus
dispatch and follow the instructions on the Emergency Response Guide.
Decontamination & Disposal Procedures
Dispose of all ethidium bromide waste through the campus Hazardous Waste
Department. Disposal of ethidium bromide solutions down the drain is NOT ALLOWED.
Disposal of dried gels containing ethidium bromide or any contaminated solid waste
(gloves, microfuge tubes, etc.) in the general campus waste stream is NOT ALLOWED.
Collection and detoxification of ethidium bromide solutions for drain disposal is NOT
ALLOWED.
Decontamination of Spills
Detoxification and decontamination of ethidium bromide by oxidation with household
bleach previously recommended has shown that this produces compounds that are
more hazardous than the original ethidium bromide compound. Therefore, OXIDATION
WITH BLEACH IS NOT AN ACCEPTABLE DESTRUCTION TECHNIQUE and is not to
be used.
Method 1 (Lunn and Sansone 1989) Recommended technique for smaller, isolated
spills of dilute solutions.
1. Turn off all electrical equipment before decontamination. Wear appropriate protective
equipment including gloves, lab coat, and safety goggles.
2. Absorb all free liquid from the spill with paper towels. Caution: The towels may
contain levels of concentrated ethidium bromide and should be handled appropriately.
3. Scrub the contaminated surface six separate times, each time using a fresh, wet
paper towel. Place all towels into a container. Collect all paper towels and debris
involved with the clean up and dispose of as hazardous waste through EH&S.
4. Dry the equipment or surface and place into service.
Contact EH&S for larger, more diffuse spills.
2.4. Gas cylinders
Compressed gas cylinders are routinely used without incident; however they pose a
severe safety hazard if handled incorrectly.
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Do not handle cylinders unless you have been properly trained.
Move cylinders only with a suitable hand-truck.
Do NOT roll or drop cylinders or let them bang against each other.
Do not move or handle a cylinder unless it is protected by a protective cap.
Leave caps on until gas is about to be used.
Always secure cylinders with a chain or strap positioned around the upper third of
the cylinder.
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Clear cylinder valves of dust and debris before attaching the correct regulator.
Only use the correct regulator for a given gas – do not mix and match!
Do not force connection fittings and never tamper with safety devices in cylinder
valves or regulators.
Stand to the side of the regulator when opening the cylinder valve.
Open valves SLOWLY!
Ensure contents of cylinders are properly identified.
Do not accept unidentified cylinders and don't rely on color codes; read the label.
Do not destroy or remove identification tags or labels.
Store cylinders in a well-ventilated area away from all sources of heat or flames.
Before returning cylinder, close the valve and replace the protective cap.
Separate empty and full cylinders during storage.
Mark empty cylinders "EMPTY" or "MT".
Know safety and first-aid requirements for gases being used.
2.5. Open flame, Bunsen burners, natural gas
Safety guidelines for open flames and Bunsen burners are simple: ensure that the area
is free of flammable items or chemicals, and be sure you know where the gas shutoff is.
Natural gas piped into the lab contains an odorant (thiol compound to provide a first line
of detection for a leak). If you smell sulfur or a smell like rotting eggs, immediately try to
locate the leak, or contact facilities management.
2.6. One Hand Scoop Method of Recapping Sharps
After use, sharps should be disposed of in the specified sharps bin. Needles should not
be recapped unless you have received documented training on using the one-hand
scoop method.
With the cap lying on the bench and the cap opening facing you, use one hand ONLY to
insert the needle into the cap. Once the cap is on, you may use the other hand to
secure the cap. Do not bend or break the needle as this could cause a puncture wound.
Discard the entire needle and syringe assembly in the sharps container.
2.7. Solvents
Solvents must always be used in the fume hood. No bench work is permitted! Ensure
that solvents are stored in the flammables cabinet when not in use. Note that any time
you are transporting a solvent; you must use the appropriate secondary-containment
carrying container. [Insert] location details.
2.8. Sonication
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Sonicators are high frequency sound generators used to disrupt cells or homogenize
samples. The two main safety concerns for sonication are hearing damage and the
creation of aerosols. When possible, sonication should be performed in the fume hood,
or with the operator wearing an aerosol-resistant mask and other PPE. When the
sonicator is in use, the operator and everyone else in the room must be provided with
appropriate ear protection. This usually includes foam ear plugs with additional “mickey
mouse ear” protection on top.
2.9. Strong Acids and Bases
All work with strong acids and bases (preparation of stock solutions, etc.) must be done
in the fume hood. The user should work with the hood shield down and wear full PPE.
This includes goggles (preferably a face shield), long pants, and a lab coat to prevent
and splattering on exposed skin. Remember to always add acid or base to water and
NOT VICE VERSA! The hydration of strong acids and bases is often very exothermic.
Be aware, and be careful. Also note that any time you are transporting a strong acid or
base, you must use the appropriate secondary-containment carrying container. [Insert]
location details.
2.10. UV
UV radiation (in the form of a 200 nm light box source) is commonly used to visualize
and cut out bands of DNA from agarose gels. When using the UV, the operator must
take precautions to cover all exposed areas of the body with UV-opaque material. A
face shield is often used – goggles are NOT sufficient! Any exposed area of the body
can sustain severe burns, and unprotected eyes can suffer permanent damage. Be
sure to wear gloves to cover your hands, and a lab coat to cover the arms and wrists. It
is your responsibility when using the UV source to warn others in the area prior to
beginning use.
2.11. Vacuum procedures
Any time you use a procedure involving pulling a vacuum, implosion protection and
shields must be in place. For example, preparing PYE media, etc. must be done in the
hood, with the preparer wearing the appropriate PPE (personal protective equipment)
and the shield of the fume hood down.
Work with vacuum pumps presents its own set of hazards. Some things to be aware of:
a) Oil vapor is extremely hazardous. Always make sure there is some sort of
filtration unit for the oil exhaust, or that there is a direct exhaust venting into the
fume hoods.
b) Make sure that pumps are always hooked into a closed system. That is, they
should not be run pumping air from the atmosphere.
c) Ensure that there is always a tray under the pump to catch any oil spills. If not
contained, this can prevent a severe slip-hazard to passersby.
2.12. Water-trap procedures
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The operator must ensure that all tubing and connections are secured! This usually
involves attaching the Tygon tubing with tie-wraps or clamps. The trap itself must be
secured as well; a ring-stand works well for this purpose.
2.13. Working with recombinant organisms
Work with recombinant organisms is governed by the National Institutes of Health (NIH)
Recombinant DNA guidelines. Prior to disposal, all recombinant organisms must be
“deactivated”. Follow the guidelines for “Liquid cultures of algae and bacteria” above,
reproduced here for convenience.
Bacterial waste under ~10 mL may be treated with an equal volume of 10% sodium
hypochlorite solution, allowed to sit for 1 hour in the hood, then disposed of in “Bleachcontaining waste”.
Algal cultures of small volumes (under 1 L) should be poured into the Waste container.
The initial culture vessel should be rinsed 2x with water, with the waste water also
transferred into the Waste container. The initial culture flask should be rinsed once with
10% sodium hypochlorite solution, with those rinsings going into the “Bleach-containing
waste”. Two more rises with water (waste going into the “Bleach-containing waste”
container) sufficiently decontaminates the flask. It may now be washed with soap and
water and put back into normal use.
Bacterial cultures of a small volume (between 10 mL and 1 L) may be treated as the
algal waste above.
When the Waste container is ¾ full, it should be autoclaved to deactivate the contents.
An autoclave cycle of 45 minutes sterilization and 45 minutes exhaust should be
sufficient. After the decontaminated solution has cooled to room temperature, it should
be poured into the Culture Biowaste Drum provided by EH&S.
Large volumes of algal or bacterial culture should be deactivated prior to disposal by
autoclaving in the original culture flask. Once cooled, the deactivated solution can also
be poured into the Culture Biowaste Drum provided by EH&S. Two rinses with water,
with rinsings going into the drum as well, are sufficient to decontaminate the flask. It
may then be washed with soap and water and will be ready for further use.
For protocols that generate culture supernatant as a result of centrifuging cell cultures,
the supernatants must be treated as a recombinant species. Small volumes (as in
plasmid minipreps) may be treated with bleach as in the first section. Larger volumes of
supernatant (as when doing a TK prep) should be poured into the 6L culture flasks
originally used to grow the strains then autoclaved. Once cool, the solution should be
poured into the EH&S provided Culture Biowaste Drum, rinsed two times as above, then
cleaned and used as usual.
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