Laboratory Safety

Christos Tsatsanis

http://molmedgp.med.uoc.gr
It's a Partnership
A safe and healthful research environment requires
that you, along with each of your colleagues,
participate actively in your institution's laboratory
safety program. It is important that you know and
follow the health, safety, and environmental
practices of your institution.
The safety program of your institution provides
essential services to help you practice safe science.
These include:

Authorizing the purchase, storage, and use of

radioisotopes.

Collection, transport, and disposal of

hazardous wastes.

Training and technical assistance.

Safety clothing: Labcoats
Protect ourselves
Protect our experiments
Safety Signs Hazard warning Signs Mandatory Signs

Danger Signs
Compressed gas Prohibition signs
signs
Emergency response

When you take a job in a new laboratory, one of the first

things you should do is ask your supervisor to review with
you the emergency response plans for the lab. Make
particular note of the locations of:

Emergency telephone numbers:

Eπείγοντα 2509
(ημέρες Γενικής Εφημερίας)
Παθολογική Κλινική 2299 (όλες τις ώρες)

Eyewash fountains and emergency showers.

Spill kits.

Emergency exits and evacuation routes.

SMOKING-Strictly Prohibited!
Fire Hazard, Health Hazard,
may affect your experiments
Fire

Fire is the most potentially devastating emergency in the

modern biology laboratory. It is imperative that you know
how to prevent fires and be prepared to respond should a
fire occur.

Preventing fires. Use of flammable solvents is a primary

cause of lab fires. Always follow these prudent practices:

Use the smallest quantities of flammable solvents

practicable.

Store stock quantities in flammables storage cabinets.

Separate flammable solvents from sources of ignition.

Never use a Bunsen burner in any area where
flammable solvents are handled.
Clothing fire! Help!

Your colleague just dropped a

250 ml beaker of alcohol that
splashed on the bench top and
the front of his lab coat. A
nearby Bunsen burner caused
the alcohol to burst into flame.
What is the first thing you
should do?
Material Safety Data Sheets

A Material Safety Data Sheet (MSDS) is a valuable

reference. It is important to consult an MSDS before
introducing a new chemical into a lab protocol or working
with hazardous substances. Prepared by its manufacturer, an
MSDS provides information to help you understand the
intrinsic hazards of the chemical including:

Physical and chemical properties.

Stability and reactivity information.
Health hazard information.
Acute and chronic effects of exposure.
Permissible exposure limits.

You should become familiar with your host institution's

Chemical Hygiene Plan or other laboratory safety
documents. You may request copies of these materials from
your supervisor or your host institution's environmental
health and safety office.
Container labels
Labels are another good
reference for information on
chemical hazards. Labels on
containers of purchased
chemicals include:

The common name of the

substance.
An appropriate hazard
warning.

Other label information may

include procedures for:

Proper handling.
Storage.
Emergency response.
 Dangerous for the
Corrosive Flammable
environment

Harmful Irritant Explosion Hazard

 Oxidizing
Toxic Very Toxic agent (O)

National Fire Protection Association's label

NFPA labels
Also valuable is the National Fire Protection
Association's labeling system that shows the type
and the degree of a chemical hazard. It is used on
some chemical containers, but is most often found
at the entrances to labs and chemical storage areas.
The labels are diamond-shaped and color-coded.

Blue indicates the health hazard.

Red indicates the fire hazard.
Yellow indicates the reactivity hazard.
White gives special information
such as water or oxidizer incompatibility.

In each field, the degree of the hazard is rated from

0 to 4, with 4 being the greatest hazard and 0
indicating no significant hazard.
Chemical storage
Safe storage of chemicals is a necessity in every laboratory.
It will:

Provide for effective management of chemicals.

Lessen the risk of fire.

Prevent accidental mixing of incompatible chemicals in

emergencies.

Minimize exposure to corrosive and toxic chemicals.

Safe chemical storage may seem to be a matter of common

sense. Yet in fact, it requires an awareness of each chemical's
potential hazards, and a lot of thought.
Rules of thumb for safe storage
Store chemicals that can ignite at room temperature in a
flammables cabinet. If flammable chemicals require cold
storage, store only in refrigerators approved for such use.

Storage areas for corrosive, toxic, flammable, and highly

reactive chemicals should be near a laboratory chemical
hood to encourage use of the hood when transferring
chemicals.

Store only cleaning materials directly under the sink.

Never store liquid hazardous chemicals above eye level.

Store heavy chemical containers on lower shelves, not

on
the floor.
Always protect your eyes
Safety glasses with side eye
shields, splash goggles, and full
face shields offer varying degrees
of protection against splattering
chemicals and airborne objects.
Choose safety glasses with side
eye shields when there is a splash
hazard with a small quantity of a
hazardous chemical, for instance,
when opening or closing a bottle or
popping open a microfuge tube.
Goggles
Wear goggles when you
are handling a chemical
that is highly caustic or in a
larger volume, perhaps a
liter or more.

Face shields
Wear a face shield when
you are handling a very
large volume of a
hazardous chemical, or
when you need to protect
your face and your eyes.
For example, wear a face
shield when you are
removing a closed
container from liquid
nitrogen or working at a
transilluminator.
Gloves
Wearing gloves is a simple
and effective way to protect
yourself from chemical
contact, but the gloves must
be resistant to the specific
chemical with which you are
working.

No glove material is
impermeable to all chemicals,
therefore, the most effective
practice in using protective
gloves is to change them
frequently and whenever they
are contaminated.
Working with chemicals
Phenol-chloroform extraction is routinely
performed in most modern biology laboratories.
Like any experiment, you should know all the
chemicals in your protocol. Although substitute
protocols that do not require phenol are available
and used in many labs, extraction with phenol-
chloroform remains the traditional approach for
purifying nucleic acids. Continued use of this
procedure accounts for the fact that phenol is the
most frequently reported chemical exposure in
laboratories.

Most labs also routinely handle acrylamide. As

with any hazardous powder, measuring and
mixing are the high-risk procedures. There are
ways to reduce the hazards of working with
acrylamide that you should know and follow.
 Toxicity- acrylamide toxicity
Major Hazards
Suspected human carcinogen (OSHA "select carcinogen") and neurotoxin.
Toxicity
The acute toxicity of acrylamide is moderate by ingestion or skin contact. Skin
exposure leads to redness and peeling of the skin of the palms. Aqueous
acrylamide solutions cause eye irritation; exposure to a 50% solution of
acrylamide caused slight corneal injury and slight conjunctival irritation, which
healed in 8 days.
The chronic toxicity of acrylamide is high. Repeated exposure to ~2 mg/kg per
day may result in neurotoxic effects, including unsteadiness, muscle
weakness, and numbness in the feet (leading to paralysis of the legs),
numbness in the hands, slurred speech, vertigo, and fatigue. Exposure to
slightly higher repeated doses in animal studies has induced multisite cancers
and reproductive effects, including abortion, reduced fertility, and mutagenicity.
Acrylamide is listed in IARC Group 2B ("possible human carcinogen") and is
classified as a "select carcinogen" under the criteria of the OSHA Laboratory
Standard.
Practicing safe science with chemicals
Working with chemicals safely means doing all the things
required for doing good science:

Know your protocol; execute procedures

meticulously.

Know your chemicals.

Use smaller quantities of chemicals; consider safer

substitute protocols.

Wear safety glasses, proper gloves, and a lab coat when

handling hazardous chemicals; avoid splash hazards.

Use a laboratory chemical hood when handling stock

quantities of volatile hazardous chemicals.
Chemical hoods
How the hood works

You can place dry ice

into a hood and see
how it actually works.
If the sash is all the
way up, the vapor
spills out of the hood
easily, compromising
protection.

When the sash is

pulled down to the
marked position,
there is better control.
The dry ice vapors
are captured readily,
increasing protection.
Laboratory Chemical Safety
Summaries

Laboratory Chemical Safety Summaries (LCSSs) are the

most relevant source of safety data for chemicals
commonly used in laboratories. Unlike an MSDS, the
information found in LCSSs applies specifically to the
lab environment. LCSSs include:

Toxicity, flammability, reactivity, and explosibility

data.
Directions for handling, storage, and disposal.
First aid and emergency response instructions.

http://www.hhmi.org/about/labsafe/index.html
http://web.princeton.edu/sites/ehs/labsafetymanual/index.html
http://www.ehs.umass.edu/lhs.html
 ETHIDIUM BROMIDE

Substance Ethidium bromide

(Dromilac, homidium bromide)
CAS 1239-45-8
Formula C21H20BrN3
Physical Properties Dark red crystals
mp 260 to 262 °C
Soluble in water (5 g/100 mL)
Odor Odorless solid
Major Hazards Potent mutagen
Toxicity Acute toxic effects from exposure to ethidium bromide have not been thoroughly investigated. Ethidium
bromide is irritating to the eyes, skin, mucous membranes, and upper respiratory tract.
Although there is no evidence for the carcinogenicity or teratogenicity of this substance in humans,
ethidium bromide is strongly mutagenic and therefore should be regarded as a possible carcinogen and
reproductive toxin.
Flammability and Explosibility Ethidium bromide does not pose a flammability hazard (NFPA rating = 1).
Reactivity and Incompatibility No incompatibilities are known.
Storage and Handling Ethidium bromide should be handled in the laboratory using the "basic prudent practices" described in
Chapter 5.C. Because of its mutagenicity, stock solutions of this compound should be prepared in a fume
hood, and protective gloves should be worn at all times while handling this substance. Operations capable
of generating ethidium bromide dust or aerosols of ethidium bromide solutions should be conducted in a
fume hood to prevent exposure by inhalation.
Accidents In the event of skin contact, immediately wash with soap and water and remove contaminated clothing. In
case of eye contact, promptly wash with copious amounts of water for 15 min (lifting upper and lower lids
occasionally) and obtain medical attention. If ethidium bromide is ingested, obtain medical attention
immediately.
In the event of a spill, mix ethidium bromide with an absorbent material (avoid raising dust), place in an
appropriate container, and dispose of properly. Soak up aqueous solutions with a spill pillow or absorbent
material.
Disposal Excess ethidium bromide and waste material containing this substance should be placed in an appropriate
container, clearly labeled, and handled according to your institution's waste disposal guidelines.
Know your radionuclides

You should know the types and energies of emitted radiations, the
potential for external exposure, the half-life, the annual limit on
intake (ALI), and how to detect contamination for the radionuclides
you use.

Phosphorus32 emits energetic beta particles

that can travel up to 6m in air. Aside from ingestion, its
principal hazard is exposure to skin and eyes.

The beta emission of sulfur35 and phosphorus33 is low

energy. Ingestion is the principal hazard.

The tritium betas have very low energy.

Ingestion is the only hazard.

Iodine125 is a gamma and x-ray emitter.

In its unbound form it presents an inhalation hazard and
can be a source for whole body exposure.
 Half-lifes and ALIs of
Common Radionuclides
Practices for reducing external
exposure

Work efficiently. Working efficiently reduces your

exposure time. Post a copy of your protocol nearby for
reference and, before you begin, assemble everything
you will need.

Keep your distance. Exposure level varies inversely

as the square of the distance from the source.
Doubling your distance from the radionuclide you are
using results in a four-fold reduction (1 / 22) in
exposure rate.

Use shielding when appropriate. Placing an acrylic

shield in the radioactive path is a good way of
reducing external exposure to the energetic beta
particles emitted by radionulcides like 32P. Shielding is
unnecessary for low energy beta emitters like 35S and
33P.
Monitoring for radiation

Use a Geiger-Müller counter to detect beta emission.

The more sensitive pancake style detector should always
be used when working with low emission isotopes
like 35S and 33P.

Use a solid scintillation detector with NaI crystal to

monitor for gamma and Bremsstrahlung radiation.

Always check new packages of radionuclides as they

arrive at the lab for possible contamination.

Before beginning a procedure, survey the work area

to be sure there is no residual contamination.
Survey the work area after completing your procedure
to make sure you do not leave behind residual
contamination.
Mammalian cell culture hazards
Tissue culture work is an important component of the modern biology
laboratory. This activity raises two concerns:

Safety of the worker because the cell line may contain an

infectious agent

Integrity of the cell cultures because contamination can ruin

the science.

The proper mindset, protective equipment, and proficiency in

experimental technique are essential to your safety and the viability of
your cell cultures.

No cell line is guaranteed to be non-hazardous

When handling mammalian blood or tissue, assume that infectious agents
are present.
The Class II Biological Safety Cabinet
The Class II Cabinet can be found in almost every
tissue culture lab. It is designed to:

Protect you from being exposed to

infectious aerosols that may be
generated within the cabinet.

Protect your cultures from microbial

contaminants that are ubiquitous in room
air.

You are protected by room air that is drawn into the

cabinet front grill and the cabinet downward airflow.
This combination of airflows quickly prevents
respirable size particles from escaping into your lab.

Your cultures are protected by the cabinet downward

airflow, which is filtered by high efficiency
particulate air (HEPA) filters. HEPA filters also treat
cabinet exhaust air.
Careful technique provides the best protection

Watch your activity. Airflow disruption affects performance.

Use slow and deliberate motions when you move your hands
into and out of the cabinet. Do not place supplies on the airflow
grills or use a Bunsen burner. A covered grill and heat
convection currents will severely disrupt airflow and increase
the risk of contamination and exposure.

Pipetting technique. Discharge pipettes against the flask or

tube wall to avoid splashes. Take great care to avoid aerosols
and splashes when mixing fluids with a pipette. Use disposable
pipettes when your tissue culture procedure involves an
infectious agent. Place used pipettes into a bleach solution
before discarding them as biohazard waste.

Handling sharps. Avoid using needles, capillary tubes,

scalpels, and other sharp instruments. When you must use them,
handle with caution to prevent punctures and cuts. Discard used
needles and disposable sharps into a puncture-resistant
container with a lid.
• Discard and Decontaminate culture fluids,
cells etc.
• Decontamination is achieved using
Clorine at a concetration of 20%
Working with human blood

Working with blood can pose

special dangers. Human blood and
other body fluids where blood is
visibly present may contain the
hepatitis B virus (HBV) or the
AIDS virus. If you handle these
materials, it is vital that you be
immunized against HBV.
Immunization will be provided at
no cost to you. Talk to your
supervisor to arrange for your
immunization.

It is important that you become

familiar with your host institution's
Exposure Control Plan. To avoid
accidental exposure to human
blood or body fluids you should
adhere to each of the following
safe work practices.
Except when drawing blood, avoid
the use of syringes, needles, and
other sharp instruments. When you
must use these things, handle them
with caution to avoid accidental
punctures or cuts.

Discard used needles and

disposable cutting instruments into
a puncture-resistant container that
has a lid. Used needles should not
be resheathed, bent, broken,
removed from disposable syringes,
or otherwise manipulated by hand.
Disinfect blood spills with a
freshly prepared one-in-ten
dilution of household bleach.
Clean all work surfaces with 70%
isopropyl alchohol when the
procedures are completed.

If you are exposed overtly to

blood, wash the exposed area or
wound with soap and water and go
to the emergency room or
occupational medical service of
your host institution. Report the
incident to your supervisor.
Physical hazards

A biomedical research laboratory,

like any other workplace, presents
a variety of physical hazards that
can be minimized by using good
laboratory practice and common
sense, by staying alert, and by
always thinking about where the
hazards are. Keep floors dry and
uncluttered to prevent slips, trips,
and falls. Whenever possible, open
flames should be replaced by
electrical heating.
3. Laboratory Damage - Cornell(note shelving torn from the wall)
Hazards
Respect the dangers of high
voltage, ultraviolet light, heat
sources, and cryogenic materials.
When transporting hazardous
substances, make certain that the
containers are sealed and use
secondary containers. Try not to
take routine manipulations for
granted, especially those involving
glass, needles, or sharps. And, if
you are fatigued or distracted, take
a break, relax and refocus. If you
notice any unsafe conditions or
have an accident or injury, talk
with your supervisor.
Centrifugation hazards
Ninety percent of centrifuge-related failures are
user errors. Careless centrifugation can mean
lost samples or damaged equipment. It can also
present a risk to the user and the lab. Fortunately
personal injury is an infrequent event. When it
occurs, it is usually associated with using
improper lifting techniques to move heavy
rotors.

Centrifugation isn't as simple as it appears. It

requires careful use, careful maintenance, and
careful bookkeeping.
1. Centrifuge Exterior - Cornell
2. Centrifuge Interior - Cornell
4. Centrifuge Interior - MIT