Laboratory Safety
Bio 181
I. General practices:
1. First and foremost, to be safe in any lab, plan your experiments well, do not hurry when
working and leave time to clean up properly.
II. Specific practices:
1. Eating and drinking are not permitted in the lab. No food or drink should be anywhere in
the lab, opened or unopened.
2. Wash your hands before beginning and after finishing work in the laboratory.
3. Long hair should be tied back when working in the laboratory. Wear closed toe shoes
whenever possible to protect your feet.
4. Never mouth pipet,
5. Disinfect your lab bench at the beginning and end of each lab.
6. Wear gloves and protective goggles or eyewear when working with toxic chemicals or
UV light.
7. Minimize clutter on your lab bench. Put away extra notebooks, books, coats, etc. so they
will not come in contact with your work. Be sure that backpacks are stored safely out the
way.
8. Clean up as you proceed through experiments and keep your work area organized.
9. Label all tubes that are in use clearly.
10. Keep hot burners and sharp objects towards the center of the bench to prevent accidental
contact.
11. Please report frayed cords or loose electrical connections to your instructor.
12. Dispose of any tubes, plates, pipet tips, etc. containing microorganisms in the biohazard
autoclave bags for sterilization. (These bags are not for materials contaminated with
chemicals—they are for microorganisms only.)
13. Dispose of broken glass and “sharps” in the containers clearly designated for these.
14. All ethidium bromide usage is to occur in the single designated area in the adjacent
laboratory.
15. Be sure you know where the following safety equipment is in the lab and how to use it:









Eyewash
Shower
Fire blanket
Fire extinguisher
First aid kit
Nearest door to exit the lab
Fire exit from the building
Nearest phones to dial for help are: my office, the stockroom, department office
or the central lobby of Sequoia hall by the elevators.
Dial 911 on any of these phones.
16. Immediately report any accident—no matter how small—to your instructor.
III. Biohazards
1. Biohazards are organisms, viruses or other biological materials that are potentially
hazardous. The other category could be defined to include blood products, plasmids and
other forms of DNA or RNA, prions, enzymes and proteins and everything else produced
by the biological world. Since most, but necessarily all, of these are destroyed by
autoclaving the standard way to handle biohazardous waste is to collect it in an
autoclavable bag and, once full, the bag is autoclaved to kill all organisms and destroy
viruses. Following this the bag may be disposed of safely.
2. The primary biohazard we will use in this course is E. coli. However, we will use strains
derived from the strain K-12. This strain of E. coli is thought to be entirely safe for
routine lab use since it is unable to survive outside the lab, unable to transfer plasmid
DNA to other bacteria and unable to infect humans. We will, however, treat everything
contaminated with live bacteria as biohazard and dispose of all of these materials in
biohazard bags, with the exception of all contaminated sharps or glass, which must be
collected in separate sharps or glass containers designated for biohazards. We also will
disinfect the lab bench at the beginning and end of each lab.
3. For safety discussion in your lab notebook: How do government regulatory agencies
define biohazards? (Cite one definition from one agency at minimum.) Do a web search
to see if you can find the latest guidelines from any agency for handling E. coli strains
derived from K12.
4. We will also be cloning mouse antibody encoding genes later in the course and inserting
these into bacterial vectors for analysis. Do any of the regulations you have found make
a distinction between working with prokaryotic and eukaryotic sequences?
IV. Physical Hazards
1. Ultraviolet (UV) light:
Ultraviolet (UV) light is a form of high energy radiation, with wavelengths just
shorter and energies just higher than the visible spectrum. In this lab we will use UV
illumination to visualize DNA stained with ethidium bromide (EtBr). EtBr is an
intercalating agent that binds to DNA and fluoresces bright orange under UV light,
allowing DNA to be visualized.
UV light damages DNA and kills cells. It is so effective in damaging DNA and cells
that it is sometimes used to “surface sterilize” laboratory benchtops between times of use.
UV light is divided into three ranges of wavelengths: near (long wave) UV (315-400
nm), midrange UV (280-315 nm), and far (short wave) UV (200-280 nm). Maximal
sensitivity in humans is about 280 nm. Exposure to direct or indirect midrange or far UV
can cause acute eye irritation with a latent period of one half to 24 hours. Because the
retina is not sensitive to UV, eye damage may result without the subject being aware of
the exposure. The latent period for feeling symptoms (sensation of sand in the eyes,
tearing, sensitivity to light, difficulty in opening eyelids, etc.) depends on the intensity of
exposure; the more intense the exposure, the shorter the latent period. Discomfort
usually disappears in 48 hours. However, it may be accompanied by a temporary
reduction in visual acuity.
In this lab we will use UV light to illuminate our gels following staining with EtBr.
For any unshielded transilluminators be sure to wear a UV protective face shield. The
smaller transilluminator we will use routinely does not require the use of a face-mask as a
magnetic safety switch prevents the UV light from being turned on unless the shield or a
camera is in place.
2. Electrophoresis:
Electrophoresis experiments are potentially hazardous because of the presence of
ionic solutions in proximity to high voltages and currents. The electrophoretic gel boxes
used in this class are designed so that the electrodes must be disconnected from the power
source before the box can be opened. However, since any surface can become
conductive, it is advisable not to touch any part of the apparatus while the unit is on.
3. Centrifugation:
Microcentrifuges and clinical centrifuges will be used in this class. We may also use
larger floor centrifuges as well. In all cases be certain that appropriate tubes or bottles
are used—containers not designed for centrifugation may shatter or collapse under the
forces generated in centrifugation. Be certain that tubes and rotors are balanced. In
larger centrifuges tubes should be weighed and balanced relative to each other. Do not
try to stop rotors manually. Be sure rotors are cleaned appropriately if spills have
occurred.
4. Gloves:
Gloves must be worn when using hazardous chemicals. Some individuals develop
allergies upon repeated exposure to certain types of gloves. This can result from either
the material used or powders applied to the glove. Inform me if you develop an allergic
reaction or know that you are allergic to gloves—we will order another type.
V. Chemical Hazards
1. MSDS (Material Safety Data Sheets) sheets are available in the lab for each chemical we
use.
2. Plasticware and chemicals:
A variety of different kinds of plastic containers are used in molecular labs. Be aware
that some plastics are severely altered by prolonged contact with certain chemicals. For
example, some types of plastics dissolve in organic solvents such as chloroform—
potentially allowing these chemicals to leak onto you! The plastic type is often indicated
on the bottom of containers with PP (polypropylene tubes being among the most
chemical and heat resistant). We will use polypropylene microcentrifuge tubes.
3. Ethidium Bromide:
Ethidium bromide (EtBr) staining is the most rapid and sensitive way of visualizing
DNA in an agarose gel. EtBr is a planar molecule that complexes with DNA by
intercalating into the double helix. When excited with UV light, EtBr fluoresces orange
allowing detection of DNA.
EtBr is a mutagen since it interferes with DNA replication if inserted into the double
helix. Because it is a mutagen it also is a potential carcinogen and must be treated with
respect. The standard concentration of EtBr used in staining gels is 1 µg/ml.
EtBr can be handled safely as long as gloves are worn when handling solutions. All
staining of gels will be handled in a designated area in the prep room and all gels
and waste will be disposed of in labeled containers. The staining solutions will be
reused many times to minimize the generation of hazardous waste. EtBr powder presents
a greater risk than solutions since it can be inhaled—no students will use EtBr powder in
this class.
To further minimize risk:
1. Solutions will be used at 1 µg/ml which is near the limit of detection of a
significant effect in mutagenicity assays.
2. As will all solutions labeling will indicate the presence of EtBr in any container in
use.
3. Always wear gloves and safety glasses or goggles when handling solutions or
stained gels.
4. Other chemicals:
We will use a number of other chemicals that are potential irritants and several others
that are possible teratogens. These will be identified at the start of each lab and any
necessary special safety precautions will be described.
References:
Bloom M, Freyer G, and Micklos D. (1995) Laboratory DNA Science. Benjamin/ Cummings
Publishing Co., Menlo Park, CA.
Lunn G and Sansone E, (1987). Ethidium bromide: destruction and decontamination of
solutions. Anal. Biochem. 162: 453-458.
Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual. 3rd edition. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.