Transcript Centrifuge Safety - College of Charleston

Biochemistry Lab Safety
1. PPE
Personal Protective Equipment: What must
be worn when you work in the laboratory.
Eye Protection
Lab Coat
Long Pants
Closed Toed Shoes – no exposed skin around feet
Lab gloves – when required
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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
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Clothing and Foot Protection
• Clothing must cover all
exposed skin including
legs/ankles
• Stockings or leggings do not
provide good coverage
• Sandals, flip-flops, Crocs,
open-toe and open-top (i.e.
ballet flat) shoes and canvas
shoes (i.e. Toms) are not
appropriate. These are not
going to protect your feet if
you drop a piece of glass with
a liquid chemical reagent in
it.
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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
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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.
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Eye Wash
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Safety Shower
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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
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are working in the hood.
In use, side-to-side panel
used as shield
Closed, not in use
✓
In use, sash (window) raised
to less than 18 inches
✓
✓
Don’t open side shields
to make one big window.
×
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• 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
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Fire Alarms –
know the location of one close to your lab
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Fire Extinguishers – we have several in
the labs and in the hallways.
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Types of Fire Extinguishers
This is a special
fire extinguisher
for combustible
metal fires. It is a
type D fire
extinguisher. You
won’t need to
use this unless
you work in a
research lab with
combustible
metals.
Most of our fire extinguishers are ABC.
It contains a dry powder to put out the
kinds of fires we might encounter in the
chemistry labs where we have class.
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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
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Keep your lab area clean.
×
Throw away used
paper towels and
used gloves,
immediately.
×
Don’t block the floor
in front of the
eyewash/shower
station.
×
Don’t leave cords
dangling because
someone will trip
over them.
×
Don’t leave things in
the floor because
someone will trip
over it.
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Once again, the number to call in an emergency is:
843-953-5611
Please take a moment now to program
this number into your cell phone.
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Centrifuge Safety
Rotor Safety
• Do not run rotors above their rated speed
• Inspect rotor for imperfections and signs of wear that can
eventually lead to catastrophic failure
• Do not drop rotor
• Rinse the rotor after every use
• Avoid using abrasive brushes for cleaning
• If you suspect rotor has been damaged, do not use it
• Do not use a rotor that is not compatible with your model
centrifuge
• Use tubes and adapters that are rated for use in the rotor
being used
Disposable tubes
Fixed angle rotor
Swinging
Bucket
rotor
Need adaptors
Accident involving improper rotor usage
Centrifuges that malfunction can create projectiles out of the
rotor shards. If the centrifuge starts to make horrible noises,
cut the power and leave the room
Loading the Centrifuge
• Be certain that tubes are balanced with a partner
• Don’t forget to include caps when weighing the
tubes for balance
• Secure the rotor on the spindle by tightening all
knobs on the lid
• Tug gently on the rotor to make sure it is secured to
the spindle
• Do not overfill bottles (3/4 full max)
Counterbalance your
labeled sample
Both knobs are tightened in some
models to secure rotor to the spindle
Unloading Centrifuge
• Take precautions if biohazards or other hazardous
material is used as aerosols can form during
vacuum cycles
• Clean the chamber from condensation and any
spills
• Never try to open the centrifuge door before the
rotor is done spinning
• Never reach a hand or anything else into the
chamber when rotor is spinning
• Note: it is sometimes difficult to look at a
spinning rotor and determine if it is spinning
Safety Overview
• http://www.youtube.com/watch?v=q_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.
Autoclave Safety
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What is an Autoclave?
An autoclave is a specialized piece of equipment
designed to deliver heat under pressure to a chamber,
with the goal of decontaminating or sterilizing the
contents of the chamber.
Personal Protective Equipment
• Autoclaves utilize steam, heat and pressure and
therefore the risk of personal injury through scalding,
burns and exploding glassware is great.
• Personal protective equipment is absolutely required.
1) Safety Glasses
2) Lab Coat
3) Long pants
4) Closed Shoes
5) Long thermal gloves
6) Face shield recommended
What can be autoclaved?
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Cultures and stocks of infectious material
Culture dishes
Tips, pipettes, gloves, paper towels, aluminum foil
Centrifuge bottles
Glassware -- all caps must be loosened
Media and other aqueous solutions
Contaminated solid items
What CANNOT be autoclaved?
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Solvents or volatiles
Chlorinated compounds (HCL, bleach)
Corrosives
Radioactive material
Some plastics
Cycle Differences
• Fluids must be autoclaved under a “liquid” setting
• Items such as pipette tips, test tubes, and
centrifuge bottles are run under “dry” or
“gravity” setting
• The difference in settings is how the cycle is
vented
• Liquids must depressurize slowly and dry cycles
conclude with a vacuum step to draw off
condensation
Loading and Unloading the Autoclave
• All screw caps must be loosened to
prevent pressure changes in the
glassware that can cause the
container to burst
• All items should be placed in an
autoclave tray to prevent scald
burns in the event of a spill
• Return autoclave trays promptly so
that other users do not skip using a
tray because they can’t find one
• Don’t skip using a tray
• Do not remove liquid that is still
boiling
• If possible, allow glassware to cool
before removing
Loosen cap by several threads
Door Safety
• Never try to open a door that is under
pressure
• Never try to speed up the venting process by
tampering with the door, by turning on and off
the machine, etc. Venting takes time.
• Know where the pressure gauges are for the
instrument you are using
• If possible, vent door slowly
Autoclaving Waste
This bag is too full
• Contaminated pipette tips and solid waste
should be sterilized prior to disposal
• Collect waste in a special autoclave-safe
biohazards bag
• Place bag in secondary container
• Vent the bag by opening
• Do not overfill bag
• After removal place entire bag in a new trash
bag so that “biohazard” signs are no longer
showing
• Sterilized waste can go into the normal trash
• Autoclave tape can be used to verify heat
delivery but it does not guarantee proper
sterilization
Container Choice
• Pyrex glass, metal, polypropylene (PP) plastic
and polycarbonate (PC) plastic are best
choices
• Polyethylene (PE), polystyrene (PS), and high
density polyethylene (HDPE) will often melt
and make a mess
Autoclaving Tips
• Add a 2 cm depth of water to trays with
glassware; the water helps eliminate air
pockets between the tray and the glass and
helps prevent glass from breaking
• Do not fill liquid past 75% volume
• Separate items to increase steam penetration
• Increase cycle time for large volumes of liquid
• Temperature must be maintained at 121°C for
at least 30 minutes
Maintenance
• Report any irregularities to your supervisor
• Do not operate if there is a steam outage
• Failed runs should be reported and logged
Overview
• http://www.youtube.com/watch?v=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.
Toxic Chemical Safety
Health Hazardous Chemicals
• Categories:
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Irritants
Carcinogens,
Acute toxicity, fatality
Sensitizers
reproductive toxins,
target organ damage
Corrosives
Carcinogens
Target Organ Effects
Reproductive Health Toxins
Irritants, sensitizers,
Corrosives
acutely toxic
Acute Toxins
Physical Health Hazards
• Common routes of exposure in the lab are inhalation
and skin absorption, while ingestion is less common.
Toxic Chemicals
• Many chemicals commonly used in the lab are toxic.
– Toxicology concerns the degree to which a chemical is hazardous
to human health.
• How do toxicologists predict which chemicals will be
toxic and determine their mechanism of action?
– Animal studies to determine a dose-response curve to predict a
threshold level above which a chemical is toxic
– Mechanistic studies to determine how a chemical will be toxic to
animals and humans
– The Ames test to assess DNA damage caused by carcinogens
– Gene microarrays to determine target genes
Toxicity and Minimizing Exposure
• Toxic reactions depend on the duration of exposure
– Acute exposure – a single exposure, or multiple exposures over 1-2 days
– Chronic exposure – multiple exposures over a longer period of time
• The Department of Labor OSHA establishes legal
permissible exposure limits (PELs) for the workplace
– “However, these standards must not be taken to represent an absolute
boundary between the positively safe and the positively unsafe.” – OSHA
website
• 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.
Carcinogens
• Genotoxic carcinogens cause DNA damage directly
(e.g., by forming a DNA adduct) and/or indirectly (e.g.,
by producing reactive oxygen species that inflict
genomic damage).
– If unrepaired before replication, DNA damage results in a
mutation.
– Mutations can result in tumor initiation if they occur in genes
related to cell division, programmed cell death, DNA repair, etc.
• Non-genotoxic carcinogens promote carcinogenesis
without damaging DNA
– For example, these chemicals might stimulate cell proliferation,
tissue invasion, or angiogenesis by binding to a receptor.
– These carcinogens mostly cause tumor promotion.
Toxic Chemicals In Biochemistry
• When working with toxic chemicals, extra attention
should be paid to selection and use of PPE.
– Protect yourself by using PPE properly and disposing
of contaminated PPE.
– Protect others by not spreading the toxic chemical
around the lab.
• Many chemicals commonly used in biochemistry are
toxic. A few common examples are detailed on the
following slides.
Ethidium Bromide
Ethidium bromide is an intercalating
agent commonly used as a fluorescent
label in molecular biology laboratories
for techniques such as agarose gel
electrophoresis.
•Avoid working with the powder,
which can be fatal when inhaled
(instead work with solutions).
•Handle in hood
•Mark areas of use and
decontaminate frequently
•Do not heat agarose with
ethidium bromide in it
•Use gloves; absorbs through skin
Acrylamide
Acrylamide is the monomeric precursor
to polyacrylamide used in SDS-PAGE.
May cause cancer. May cause heritable
genetic damage. Also toxic in contact
with skin and if swallowed. Danger of
serious damage to health by prolonged
exposure through inhalation, in contact
with skin or if swallowed.
•Avoid working with the powder due to inhalation hazard
(instead work with solutions)
•Use gloves when handling
•Polymerize excess solution for safer disposal
Sodium Azide
Sodium azide is commonly found in dilute
solutions to prevent bacterial growth.
The acute toxicity of sodium azide is high
•Do not allow sodium azide to come into contact with heavy metals or their salts,
because a reaction may form heavy metal azides, which are explosives.
•Do not allow sodium azide to come into contact with aqueous acids, because
reaction liberates highly toxic hydrazoic acid, which is a dangerous explosive.
 Containers should be stored in secondary containers in a cool, dry secured
storage area separated from acids.
 Avoid using metal spatulas
 Do not dispose of solutions down the drain, as explosions could result.
Phenylmethanesulfonyl Fluoride
PMSF is used in solution to inhibit proteases
Toxic if swallowed. Causes severe skin burns and
eye damage. Extremely destructive to tissues of
mucous membranes and respiratory tract.
Corrosive. Target Organs: Nerves, Heart, Blood,
Eyes.
•Wear gloves.
•Take extra precautions when working with the powder. Do not leave any traces of
spilled power on the bench, balance, etc., where it could endanger another lab user.
Toxic Chemicals in
Other Branches of Chemistry
• Many chemicals commonly used in other branches of
chemistry (e.g., synthetic chemistry) are also toxic.
• Some examples:
– Halogenated aliphatic hydrocarbons (e.g., chloroform,
carbon tetrachloride, etc.) – cause central nervous system
depression, liver injury, kidney injury, and some degree of
cardiotoxicity. Many are carcinogenic.
– Aromatics (e.g., benzene, toluene, xylene) – cause central
nervous system depression, skin irritation. Benzene causes
bone marrow injury and is associated with leukemia.
Toxic Chemicals and Material Safety
Data Sheets (MSDS)
• Identify toxicity hazards for any chemical by consulting the MSDS. The
Hazards section includes toxicity warnings:
Toxic Chemicals and Material Safety
Data Sheets (MSDS)
• Look for the health NFPA category rated from 0-4, and read warning
statements.
0 – Hazard no greater than ordinary material
1 – May cause irritation; minimal residual injury
2 – Intense or prolonged exposure may cause incapacitation;
residual injury may occur if not treated
3 – Exposure could cause serious injury even if treated
4 – Exposure may cause death
Toxic Chemicals and Material Safety
Data Sheets (MSDS)
• Separate sections indicate toxicological data on the chemical:
Biohazard Safety
Biological Hazards
• Biological hazards are potential sources of infectious
agents that could be harmful to human health.
– Bacterial, fungal, parasitic, viral, and prion agents.
– Sources may include animals, tissues, cells, blood, and
nucleic acid samples, including recombinant DNA.
Biohazard Classification
• The National Institutes of Health (NIH) has
determined a classification system for biohazardous
agents based on Risk Groups (RG).
Risk Group 1
(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)
Biological Hazards
• Sterilization techniques are generally effective in
destroying biohazard agents (except prions and spores)
– Autoclave
– Bleach
– Alcohol
• Biohazards are another reason (in addition to avoiding
ingestion of toxic chemicals) for good lab hygiene
– Glove use and proper gloves hygiene
– Hand washing
– No food or drink in lab
Other Biochemistry Safety
Concerns
Ultracold (-80°C) Freezer Use
• Many biological samples and chemicals need to be
preserved at temperatures below room temperature
– Always consult the label: 4°C (refrigerator), -20°C (conventional
freezer), -80°C (ultracold freezer), -196°C (liquid nitrogen)
– Cold storage can slow cell death rate, preserve enzyme activity,
inhibit contaminating bacterial growth, and prevent
degradation.
– Use insulated gloves to handle ultracold materials.
– Handle glass dewars with caution – danger of
exploding glass if they are knocked over and broken.
High Voltage Techniques
• DNA and protein gel electrophoresis combine high
voltage with the use of aqueous solutions
– Take precautions to avoid electrocution.
– Modern gel boxes have electrodes positioned on the lids to
drastically reduce the risk of electrocution.
– Always secure the gel box lid before turning on the voltage.
Turn off the voltage before removing the lid to a gel box.
– Match the red and black electrodes to the corresponding red
and black outlets on the power supply.
GHS Symbols
Liquid Nitrogen Safety
• Liquid nitrogen (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
Liquid Nitrogen DON’T’s
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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!
Liquid Nitrogen Do’s
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
NO!
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
YES!
• Closed toed Shoes
• Long pants
Special Note on LN2 transport for Chemistry Magic Shows
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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