Lab Safety 3

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 signs

Prohibition 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.

Corrosive

Harmful

Flammable

Irritant

Dangerous for the environment

Explosion Hazard

Toxic

Very Toxic

National Fire Protection Association's label

Oxidizing agent (O)

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 phenolchloroform 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

Formula Physical Properties

Odor Major Hazards Toxicity

Ethidium bromide (Dromilac, homidium bromide) CAS 1239-45-8 C21H20BrN3 Dark red crystals mp 260 to 262 °C Soluble in water (5 g/100 mL) Odorless solid Potent mutagen 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